Compositions and methods for treating lung disease and injury

ABSTRACT

Disclosed herein are therapeutic methods for treating lung diseases, disorders and injury in a mammal, including treatment of acute respiratory distress syndrome (ARDS), acute lung injury, pulmonary fibrosis (idiopathic), bleomycin induced pulmonary fibrosis, mechanical ventilator induced lung injury, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiolitis obliterans after lung transplantation and lung transplantation-induced acute graft dysfunction, including treatment, prevention or prevention of progression of primary graft failure, ischemia-reperfusion injury, reperfusion injury, reperfusion edema, allograft dysfunction, pulmonary reimplantation response, bronchiolitis obliterans after lung transplantation and/or primary graft dysfunction (PGD) after organ transplantation, in particular in lung transplantation, comprising down-regulating the TLR2 gene or both the TLR2 gene and TLR4 gene. Provided herein are compositions, methods and kits for treating lung diseases, disorders and injury.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/448,723, filed Mar. 3, 2011, entitled “Combination Therapy forTreating Lung Disease And Injury” and which is incorporated herein byreference in its entirety and for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which is entitled228-PCT1_ST25.txt, created on Feb. 6, 2012 and 2,280 kb in size, and ishereby incorporated by reference in its entirety.

Throughout this application various patents and publications are cited.The disclosures of these documents in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

FIELD OF THE INVENTION

Compositions, methods and kits for treating lung disease and injury areprovided herein.

SUMMARY OF THE INVENTION

Compositions, methods and kits for treating lung diseases are providedherein. In certain aspects and embodiments, provided are compositionsand methods for therapy for treating lung disorders or injury in amammal, including treatment of acute respiratory distress syndrome(ARDS), acute lung injury, pulmonary fibrosis (idiopathic), bleomycininduced pulmonary fibrosis, mechanical ventilator induced lung injury,chronic obstructive pulmonary disease (COPD), chronic bronchitis,emphysema, lung transplantation-induced acute graft dysfunction andbronchiolitis obliterans after lung transplantation. In certain aspectsand embodiments, provided are compositions and methods for combinationtherapy for treating or preventing inflammation and/or graft rejectionassociated with organ transplantation, in particular lungtransplantation, including treatment, prevention or attenuation ofprogression of primary graft failure, ischemia-reperfusion injury,reperfusion injury, reperfusion edema, allograft dysfunction, pulmonaryreimplantation response, bronchiolitis obliterans after lungtransplantation and/or primary graft dysfunction (PGD) after organtransplantation, in particular in lung transplantation. In certainaspects and embodiments, provided are compositions and methods forcombination therapy for treating lung disorders or injury in a mammal.The compositions and methods involve inhibiting the gene Toll-likereceptor 2 (TLR2) or the genes Toll-like receptor 2 (TLR2) and Toll-likereceptor 4 (TLR4).

In various aspects and embodiments, compositions, methods and kitsprovided herein may target, decrease, down-regulate or inhibit theexpression/activity/function of the gene Toll-like receptor 2 (TLR2). Invarious aspects and embodiments, compositions, methods and kits providedherein may target, decrease, down-regulate or inhibit theexpression/activity/function of the genes: (i) Toll-like receptor 2(TLR2) and (ii) Toll-like receptor 4 (TLR4).

In one aspect, provided is a method for treating a lung disorder,disease or injury in a mammal in need thereof. The method may includeadministering to the mammal at least one therapeutic agent selected froma TLR2 inhibitor or a pharmaceutically acceptable salt or prodrugthereof, in an amount effective to treat the mammal.

In another aspect, provided is a method for treating a lung disorder,disease or injury in a mammal in need thereof. The method may includeadministering to the mammal at least two therapeutic agents selectedfrom: (i) a TLR2 inhibitor or a pharmaceutically acceptable salt orprodrug thereof, and (ii) a TLR4 inhibitor or a pharmaceuticallyacceptable salt or prodrug thereof; in an amount effective to treat themammal.

The methods may include preventing, treating, ameliorating, and/orslowing the progression of lung disorders or injury, such as, withoutbeing limited to, ARDS, acute lung injury, pulmonary fibrosis(idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilator induced lung injury, COPD and disease, disorder or injuryassociated with lung transplantation in a subject. The method mayinvolve treating, ameliorating, and/or slowing the progression of theaforementioned diseases or conditions or associated symptoms orcomplications thereof by administering to said subject a therapeuticallyeffective amount of a therapeutic agent directed to the gene TLR2. Themethod may involve treating, ameliorating, and/or slowing theprogression of the aforementioned diseases or conditions or associatedsymptoms or complications thereof by administering to said subject atherapeutically effective amount of at least one therapeutic agent thatdown regulates TLR2 and at least one therapeutic agent that downregulates TLR4. The method may involve treating, ameliorating, and/orslowing the progression of the aforementioned diseases or conditions orassociated symptoms or complications thereof by administering to saidsubject a therapeutically effective amount of a single therapeuticagent, which is capable of down-regulating the genes TLR2 and TLR4and/or the gene products of the genes TLR2 and TLR4.

In various embodiments the provided methods of treating a lung disease,disorder or injury comprise inhibiting the gene Toll-like receptor 2(TLR2) in combination with one or more additional treatment methodsselected from the group consisting of surgery, steroid therapy,non-steroid therapy, antibiotic therapy, antiviral therapy, antifungaltherapy, immunosuppressant therapy, anti-infective therapy,anti-hypertensive therapy and nutritional supplements. In variousembodiments the additional treatment is administered prior to,subsequent to or concomitantly with the provided method for treating alung disorder, disease or injury. In various embodiments the providedmethods of treating a lung disease, disorder or injury compriseinhibiting the gene Toll-like receptor 2 (TLR2) in combination withimmunosuppressant therapy. In various embodiments the provided methodsof treating a lung disease, disorder or injury comprise inhibiting thegenes Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) incombination with one or more additional treatment methods selected fromthe group consisting of surgery, steroid therapy, non-steroid therapy,antibiotic therapy, antiviral therapy, antifungal therapy, antimicrobialtherapy, immunosuppressant therapy, anti-infective therapy,anti-hypertensive therapy and nutritional supplements. In variousembodiments the provided methods of treating a lung disease, disorder orinjury comprise down-regulating the gene Toll-like receptor 2 (TLR2) andthe gene Toll-like receptor 4 (TLR4) in combination withimmunosuppressant therapy.

In certain embodiments the provided methods may include one or more ofthe following:

A. Administration of a pharmaceutical composition comprising atherapeutic agent selected from a TLR2 inhibitor or a pharmaceuticallyacceptable salt or prodrug thereof; and a pharmaceutically acceptablecarrier; or

B. Co-administration, e.g. concomitantly or in sequence, of atherapeutically effective amount of at least two therapeutic agents,wherein at least one therapeutic agent is for down-regulating the geneTLR2 and at least one therapeutic agent is for down-regulating the geneTLR4; and the therapeutic agents are selected from: (i) a TLR2 inhibitoror a pharmaceutically acceptable salt or prodrug thereof, and (ii) aTLR4 inhibitor or a pharmaceutically acceptable salt or prodrug thereof;or

C. Administration of a pharmaceutical composition comprising acombination of at least two therapeutic agents, wherein at least onetherapeutic agent is for down-regulating the gene TLR2 and at least onetherapeutic agent is for down-regulating the gene TLR4; and thetherapeutic agents are selected from: (i) a TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof, and (ii) a TLR4inhibitor or a pharmaceutically acceptable salt or prodrug thereof; anda pharmaceutically acceptable carrier; or

D. Administration of a pharmaceutical composition comprising atherapeutic agent which is capable of down-regulating the genes TLR2 andTLR4 and/or the gene products of the genes TLR2 and TLR4. Non-limitingexamples of such single agents are tandem and multi-armed RNAi moleculesdisclosed in PCT Patent Publication No. WO 2007/091269.

In one aspect, provided is a medicament that includes a therapeuticagent which target, decrease, down-regulate or inhibit theexpression/activity/function of the gene TLR2, or a pharmaceuticallyacceptable salt or prodrug thereof. Therapeutic agents useful in thecombination as provided herein include, but are not limited to, smallorganic molecule chemical compounds; proteins, antibodies or fragmentsthereof, peptides, peptidomimetics and nucleic acid molecules.

In another aspect, provided is a medicament that includes at least twotherapeutic agents which target, decrease, down-regulate or inhibit theexpression/activity/function of the genes: (i) TLR2 and (ii) TLR4,wherein at least one therapeutic agent down-regulates the gene TLR2 andat least one therapeutic agent down-regulates the gene TLR4; and thetherapeutic agents are selected from: (i) a TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof, and (ii) a TLR4inhibitor or a pharmaceutically acceptable salt or prodrug thereof.Therapeutic agents useful in the combination as provided herein include,but are not limited to, small organic molecule; proteins, antibodies orfragments thereof, peptides, peptidomimetics and nucleic acid molecules.

In some embodiments the therapeutic agent comprises a nucleic acidmolecule. In some embodiments each nucleic acid molecule isindependently selected from the group consisting of an antisensemolecule, a short interfering nucleic acid (siNA), short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA) or short hairpinRNA (shRNA) that bind a nucleotide sequence (such as an mRNA sequence)encoding a target gene selected from TLR2 and TLR4, for example:

-   -   the mRNA coding sequence for human TLR2 exemplified by SEQ ID        NO:1 (gi|68160956|ref|NM_003264.3| Homo sapiens toll-like        receptor 2 (TLR2), mRNA), or    -   the mRNA coding sequence for human TLR4 exemplified by SEQ ID        NO:2 (gi|207028550|ref|NR_024169.1| Homo sapiens toll-like        receptor 4 (TLR4), transcript variant 4, non-coding RNA); or    -   the mRNA coding sequence for human TLR4 exemplified by SEQ ID        NO:3 (gi|207028620|ref|NM_138554.3| Homo sapiens toll-like        receptor 4 (TLR4), transcript variant 1, mRNA); or    -   the mRNA coding sequence for human TLR4 exemplified by SEQ ID        NO:4 (gi|207028451|ref|NR_024168.1| Homo sapiens toll-like        receptor 4 (TLR4), transcript variant 3, non-coding RNA).

In various embodiments each nucleic acid molecule is or includes a dsRNAmolecule or a siRNA molecule. In various embodiments, the nucleic acidmolecule (a) includes a sense strand and an antisense strand; (b) eachstrand of the nucleic acid molecule is independently 17 to 40nucleotides in length; (c) a 17 to 40 nucleotide sequence of theantisense strand is complementary to a sequence of an mRNA encodinghuman TLR2 (e.g., SEQ ID NO: 1) or TLR4 (e.g., SEQ ID NOs: 2-4); and (d)a 17 to 40 nucleotide sequence of the sense strand is complementary tothe a sequence of the antisense strand and includes a 17 to 40nucleotide sequence of an mRNA encoding human TLR2 (e.g., SEQ ID NO: 1)or TLR4 (e.g., SEQ ID NOs: 2-4).

A pharmaceutical product as provided herein may, for example, be apharmaceutical composition including the therapeutic agent in apharmaceutically acceptable carrier. A pharmaceutical product asprovided herein may, for example, be a pharmaceutical compositionincluding the first and second therapeutic agent in admixture in apharmaceutically acceptable carrier. Alternatively, the pharmaceuticalproduct may, for example, be a kit comprising a preparation of the firsttherapeutic agent and a preparation of the second therapeutic agent and,optionally, instructions for the simultaneous, sequential or separateadministration of the preparations to a patient in need thereof.

In a first aspect, provided is a method of preventing or reducing thesymptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, comprising administering to the recipient atherapeutically-effective amount of at least one TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof, and atherapeutically-effective amount of at least one TLR4 inhibitor or apharmaceutically acceptable salt or prodrug thereof, thereby preventingor reducing the symptoms of PGD in the recipient. In various embodimentsthe symptoms of PGD include inflammation, acute graft rejection, graftrejection, ischemia-reperfusion injury, reperfusion injury, impairedpulmonary function, bronchiolitis obliterans, impaired bloodoxygenation, increased inflammatory cytokine production, intra-graft andintra-airway accumulation of granulocytes, pulmonary edema andhypoxemia.

In some embodiments, the recipient of the lung transplant is a humanthat is at risk of developing or is being treated for primary graftdysfunction (PGD). In some embodiments the method as provided hereinmay, for example, be use for preventing or reducing the symptoms of coldischemia-associated PGD. Alternatively, the method may, for example, befor preventing or reducing the symptoms of warm ischemia-associated PGD.

In various embodiments, the administration of the at least oneTLR2inhibitor or a pharmaceutically acceptable salt or prodrug thereof, andthe at least one TLR4 inhibitor or a pharmaceutically acceptable salt orprodrug thereof results in one or more of the following: reducedpulmonary edema, increased blood oxygenation, preserved bloodoxygenation, improved pulmonary function, preserved pulmonary functionin the recipient of a lung transplant and improved pulmonary function ofthe transplanted lung.

In various embodiments, the at least one TLR2 inhibitor and the at leastone TLR4 inhibitor are administered to the recipient of a lungtransplant prior to, during or following the lung transplantation.

In some embodiments, the at least one TLR2 inhibitor and the at leastone TLR4 inhibitor are co-administered to the recipient in the sameformulation. Alternatively, the at least one TLR2 inhibitor and the atleast one TLR4 inhibitor are co-administered to the recipient indifferent formulations.

In some embodiments, the at least one TLR2 inhibitor and the at leastone TLR4 inhibitor are co-administered to the recipient by the sameroute. In other embodiments, the at least one TLR2 inhibitor and the atleast one TLR4 inhibitor are co-administered to the recipient bydifferent routes. In various embodiments, the methods comprisesimultaneous administration of the at least one TLR2 inhibitor and theat least one TLR4 inhibitor. In some embodiments, the methods compriseseparate administration of the at least one TLR2 inhibitor and the atleast one TLR4 inhibitor. In some embodiments, the methods comprisecombined administration of the at least one TLR2 inhibitor and the atleast one TLR4 inhibitor. In other embodiments, the methods comprisesequential administration of the at least one TLR2 inhibitor and the atleast one TLR4 inhibitor.

In various embodiments the provided method of preventing or reducing thesymptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, further comprises at least one additional treatment selectedfrom the group consisting of surgery, steroid therapy, non-steroidtherapy, antiviral therapy, antifungal therapy, antimicrobial therapy,immunosuppressant therapy, anti-infective therapy, anti-hypertensivetherapy, nutritional supplements and any combination thereof. In variousembodiments, the additional treatment is administered prior to,subsequent to or concomitantly with administering of at least one TLR2inhibitor and at least one TLR4 inhibitor. In some embodiments, theadditional treatment comprises immunosuppressant therapy.

In various embodiments, the route of administration of at least one TLR2inhibitor and at least one TLR4 inhibitor is selected from: systemicadministration or local administration. In various embodiments, themethod of administration of at least one TLR2 inhibitor and at least oneTLR4 inhibitor to the recipient of a lung transplant is selected fromthe group comprising: intravenous, intraarterial, intraperitoneal,intramuscular, intraportal, subcutaneous, direct injection,intratracheal instillation, inhalation, intranasal, pulmonary andadministration via pump into the lung. In some embodiments, at least oneTLR2 inhibitor and at least one TLR4 inhibitor are administered to therecipient of a lung transplant by inhalation. In another embodiments, atleast one TLR2 inhibitor and at least one TLR4 inhibitor areadministered to the recipient of a lung transplant by intratrachealinstillation.

In various embodiments of the provided method of preventing or reducingthe symptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, the at least one TLR2 inhibitor and the at least one TLR4inhibitor are each independently selected from the group consisting of asmall organic molecule, a protein, an antibody or fragment thereof, apeptide, a peptidomimetic and a nucleic acid molecule. In someembodiments, at least one inhibitor comprises a nucleic acid molecule.In other embodiments, each inhibitor comprises a nucleic acid molecule.In some embodiments, each inhibitor comprises a nucleic acid moleculeand the first nucleic acid molecule is a double-stranded oligonucleotidethat binds a nucleotide sequence encoding a TLR2 gene and the secondnucleic acid molecule is a double-stranded oligonucleotide that binds anucleotide sequence encoding a TLR4 gene. In some embodiments thedouble-stranded oligonucleotides are linked one to the other in tandemor annealed in RNAistar formation.

In some embodiments the first double-stranded oligonucleotide comprises:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR2; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand;        and the second double-stranded oligonucleotide comprises:    -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR4; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In various embodiments the mRNA polynucleotide sequence of TLR2 is setforth in SEQ ID NO:1 and the mRNA polynucleotide sequence of TLR4 is setforth in any one of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

In some embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are co-administered to therecipient in the same formulation. In other embodiments, the firstdouble-stranded oligonucleotide and the second double-strandedoligonucleotide are co-administered to the recipient in differentformulations. In some embodiments, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areco-administered to the recipient by the same route. In some embodiments,the first double-stranded oligonucleotide and the second double-strandedoligonucleotide are co-administered to the recipient by differentroutes. In various embodiments the mode of administration of the firstdouble-stranded oligonucleotide and the second double-strandedoligonucleotide to the recipient of the lung transplant is selected fromthe group comprising: separate, combined, simultaneous and sequentialadministration.

In some embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are formulated for administeringto the recipient once. In other embodiments, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areformulated for administering to the recipient at least once-a-day. Inyet other embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are formulated for multipleadministrations to the recipient.

In some embodiments of the provided method of preventing or reducing thesymptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, at least one double-stranded oligonucleotide in dependentlycomprises a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y; wherein each of x and yis independently an integer between 17 and 40;wherein the sequence of (N′)y is complementary to the sequence of (N)x;and wherein (N)x comprises an antisense sequence to an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4.

In various embodiments of structure (A1), the mRNA polynucleotidesequence of TLR2 is set forth in SEQ ID NO:1 and the mRNA polynucleotidesequence of TLR4 is set forth in any one of SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:4.

In some preferred embodiments of structure (A1), x=y=19.

In some embodiments of structure (A1), (N)x comprises an antisenseoligonucleotide selected from the group consisting of oligonucleotideshaving SEQ ID NOs: 723-1440, 2247-3052, 7076-8312 and 8459-8604 and(N′)y comprises a complementary sense strand oligonucleotide selectedfrom the group consisting of oligonucleotides having SEQ ID NOs: 5-722,1441-2246, 5839-7075 and 8313-8458.

In various embodiments of the provided method of preventing or reducingthe symptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, administration of the at least one double-strandedoligonucleotide that binds a nucleotide sequence encoding a TLR2 geneand the at least one double-stranded oligonucleotide that binds anucleotide sequence encoding a TLR4 gene results in down-regulation ofTLR2 expression and TLR4 expression, respectively.

In some embodiments of the provided method of preventing or reducing thesymptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, at least one double-stranded compound independentlycomprises a structure (A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′(sense strand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand wherein (N)x is complementary to a consecutive sequence in an mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is covalently bound to (N)x and is mismatched to the mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of structure (A2), the mRNA polynucleotidesequence of TLR2 is set forth in SEQ ID NO:1 and the mRNA polynucleotidesequence of TLR4 is set forth in any one of SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:4.

In some preferred embodiments of structure (A2), x=y=18.

In some embodiments of structure (A2), the sequence of (N)x comprises anantisense strand oligonucleotide selected from the group consisting ofoligonucleotides having SEQ ID NOs: 4153-5252, 5546-5838, 10319-12032,and 12085-12136 and the sequence of (N′)y comprises a sense strandoligonucleotide selected from the group consisting of oligonucleotideshaving SEQ ID NOs: 3053-4152, 5253-5545, 8605-10318, and 12033-12084.

In a second aspect, provided is a method for treating a lung disorder,disease or injury in a patient in need thereof comprising administeringto the patient a therapeutically-effective combination of at least oneTLR2 inhibitor or a pharmaceutically acceptable salt or prodrug thereof,and at least one TLR4 inhibitor or a pharmaceutically acceptable salt orprodrug thereof, thereby treating the lung disorder, disease or injuryin the patient. In various embodiments, the lung disorder, disease orinjury is selected from acute respiratory distress syndrome (ARDS),acute lung injury, pulmonary fibrosis (idiopathic), bleomycin inducedpulmonary fibrosis, mechanical ventilator induced lung injury, chronicobstructive pulmonary disease (COPD), chronic bronchitis, a disorderassociated with lung transplantation and emphysema. In some embodiments,the lung disorder, disease or injury is a disorder associated with lungtransplantation. In various embodiments, the lung disorder associatedwith lung transplantation is selected from the group consisting ofinflammation, graft rejection, primary graft failure,ischemia-reperfusion injury, reperfusion injury, reperfusion edema,allograft dysfunction, acute graft dysfunction, pulmonary reimplantationresponse, bronchiolitis obliterans and primary graft dysfunction (PGD).In one embodiment, the lung disorder associated with lungtransplantation is PGD.

In some embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, the at least one TLR2inhibitor and the at least one TLR4 inhibitor are co-administered to therecipient in the same formulation. In other embodiments, the at leastone TLR2 inhibitor and the at least one TLR4 inhibitor areco-administered to the recipient in different formulations. In variousembodiments, the at least one TLR2 inhibitor and the at least one TLR4inhibitor are co-administered to the recipient by the same route. Inother embodiments, the at least one TLR2 inhibitor and the at least oneTLR4 inhibitor are co-administered to the recipient by different routes.In various embodiments the mode of administration of the at least oneTLR2 inhibitor and the at least one TLR4 inhibitor is selected from thegroup comprising: separate, combined, simultaneous and sequentialadministration.

In some embodiments, the provided method for treating a lung disorder,disease or injury in a patient in need thereof, further comprises atleast one additional treatment selected from the group consisting ofsurgery, steroid therapy, non-steroid therapy, antiviral therapy,antifungal therapy, antimicrobial therapy, immunosuppressant therapy,anti-infective therapy, anti-hypertensive therapy, nutritionalsupplements and any combination thereof. In some embodiments, theadditional treatment comprises immunosuppressant therapy. In variousembodiments, the additional treatment is administered prior to,subsequent to or concomitantly with administering of at least one TLR2inhibitor and at least one TLR4 inhibitor.

In some embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, the administering of atleast one TLR2 inhibitor and at least one TLR4 inhibitor to the patientcomprises systemic administration or local administration. In variousembodiments the method of administration is selected from the groupcomprising intravenous, intraarterial, intraperitoneal, intramuscular,intraportal, subcutaneous, direct injection, intratrachcal instillation,inhalation, intranasal, pulmonary and administration via pump into thelung. In some embodiments, the method of administration comprisesinhalation. In some embodiments, the method of administration comprisesintratracheal instillation.

In some embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, the at least one TLR2inhibitor and the at least one TLR4 inhibitor are each inhibitor isindependently selected from the group consisting of a small organicmolecule, a protein, an antibody or fragment thereof, a peptide, apeptidomimetic and a nucleic acid molecule. In some embodiments, atleast one inhibitor comprises a nucleic acid molecule. In otherembodiments, each inhibitor comprises a nucleic acid molecule. Invarious embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, a first nucleic acidmolecule is a double-stranded oligonucleotide that binds a nucleotidesequence encoding a TLR2 gene and a second nucleic acid molecule is adouble-stranded oligonucleotide that binds a nucleotide sequenceencoding a TLR4 gene. In some embodiments, the double-strandedoligonucleotides are linked one to the other in tandem or annealed inRNAistar formation.

In some embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, the firstdouble-stranded oligonucleotide comprises:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR2; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand;        and the second double-stranded oligonucleotide comprises:    -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR4; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In various embodiments the mRNA polynucleotide sequence of TLR2 is setforth in SEQ ID NO:1 and the mRNA polynucleotide sequence of TLR4 is setforth in any one of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

In some embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are co-administered to thepatient in the same formulation. In other embodiments, the firstdouble-stranded oligonucleotide and the second double-strandedoligonucleotide are co-administered to the patient in differentformulations. In some embodiments, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areco-administered to the patient by the same route. In some embodiments,the first double-stranded oligonucleotide and the second double-strandedoligonucleotide are co-administered to the patient by different routes.In various embodiments, the mode of administration of the firstdouble-stranded oligonucleotide and the second double-strandedoligonucleotide to the recipient of the lung transplant is selected fromthe group comprising: separate, combined, simultaneous and sequentialadministration.

In some embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are formulated for administeringto the patient once. In other embodiments, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areformulated for administering to the patient at least once-a-day. Inother embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are formulated for multipleadministrations to the patient.

In some embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, at least onedouble-stranded oligonucleotide comprises a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y; wherein each of x and yis independently an integer between 17 and 40;wherein the sequence of (N′)y is complementary to the sequence of (N)x;and wherein (N)x comprises an antisense sequence to an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4.

In various embodiments of structure (A1), the mRNA polynucleotidesequence of TLR2 is set forth in SEQ ID NO:1 and the mRNA polynucleotidesequence of TLR4 is set forth in any one of SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:4.

In some preferred embodiments of structure (A1), x=y=19.

In some embodiments of structure (A1), (N)x comprises an antisenseoligonucleotide selected from the group consisting of oligonucleotideshaving SEQ ID NOs: 723-1440, 2247-3052, 7076-8312 and 8459-8604 and(N′)y comprises a sense strand oligonucleotide selected from the groupconsisting of oligonucleotides having SEQ ID NOs: 5-722, 1441-2246,5839-7075 and 8313-8458.

In some embodiments of the provided method for treating a lung disorder,disease or injury in a patient in need thereof, at least onedouble-stranded compound comprises a structure (A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′(sense strand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand wherein (N)x is complementary to a consecutive sequence in an mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is covalently bound to (N)x and is mismatched to the mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of structure (A2), the mRNA polynucleotidesequence of TLR2 is set forth in SEQ ID NO:1 and the mRNA polynucleotidesequence of TLR4 is set forth in any one of SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:4.

In some preferred embodiments of structure (A2), x=y=18.

In some embodiments of structure (A2), the sequence of (N)x comprises anantisense oligonucleotide selected from the group consisting ofoligonucleotides having SEQ ID NOs: 4153-5252, 5546-5838, 10319-12032,and 12085-12136 and the sequence of (N′)y comprises a senseoligonucleotide selected from the group consisting of oligonucleotideshaving SEQ ID NOs: 3053-4152, 5253-5545, 8605-10318, and 12033-12084.

In another aspect, provided is a composition comprising at least oneTLR2 inhibitor or a pharmaceutically acceptable salt or prodrug thereofand at least one TLR4 inhibitor or a pharmaceutically acceptable salt orprodrug thereof; and a pharmaceutically acceptable carrier. In variousembodiments, each inhibitor is independently selected from the groupconsisting of a small organic molecule; a protein, an antibody orfragments thereof, a peptide, a peptidomimetic and a nucleic acidmolecule. In some embodiments, each inhibitor is independently selectedfrom the group consisting of a small organic molecule; a protein; anantibody or fragment thereof; and a nucleic acid molecule.

In some embodiments of the provided composition, each inhibitorcomprises a nucleic acid molecule. In some embodiments a first nucleicacid molecule is a double-stranded oligonucleotide that binds anucleotide sequence encoding a TLR2 gene and a second nucleic acidmolecule is a double-stranded oligonucleotide that binds a nucleotidesequence encoding a TLR4 gene. In some embodiments of the compositionthe nucleic acid molecules are linked in tandem or annealed in RNAistarformation.

In some embodiments of the provided composition, a first double-strandedoligonucleotide comprises:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR2; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand;        and a second double-stranded oligonucleotide comprises:    -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR4; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In various embodiments the mRNA polynucleotide sequence of TLR2 is setforth in SEQ ID NO:1 and the mRNA polynucleotide sequence of TLR4 is setforth in any one of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

In some embodiments, the amount of each double-stranded oligonucleotidein the composition independently ranges from about 0.05 mg to about 10.0mg.

In some embodiments of the provided composition, at least onedouble-stranded oligonucleotide independently comprises a structure(A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and

wherein (N)x comprises an antisense sequence to an mRNA selected from anmRNA encoding TLR2 and an mRNA encoding TLR4.

In various embodiments of the composition, in structure (A1), the mRNApolynucleotide sequence of TLR2 is set forth in SEQ ID NO:1 and the mRNApolynucleotide sequence of TLR4 is set forth in any one of SEQ ID NO:2,SEQ ID NO:3 and SEQ ID NO:4.

In some preferred embodiments of the composition, in structure (A1),x=y=19.

In some embodiments of the composition, at least one double-strandedoligonucleotide compound independently comprises a structure (A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′(sense strand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of the provided composition, in structure (A2),the mRNA polynucleotide sequence of TLR2 is set forth in SEQ ID NO:1 andthe mRNA polynucleotide sequence of TLR4 is set forth in any one of SEQID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

In some preferred embodiments of the provided composition, in structure(A2), x=y=18.

In some embodiments, the provided composition is formulated foradministering to the recipient once. In other embodiments, the providedcomposition is formulated for administering to the recipient at leastonce-a-day. In yet other embodiments, the provided composition isformulated for multiple administrations to the recipient.

In another aspect, provided is a kit comprising at least two therapeuticagents, wherein at least one agent comprises a TLR2 inhibitor and asecond agent comprises a TLR4 inhibitor; optionally with instructionsfor use.

In some embodiments of the provided kit, each therapeutic agent isindependently selected from the group consisting of a small organicmolecule, a protein, an antibody or fragment thereof, a peptide, apeptidomimetic and nucleic acid molecule. In some embodiments of thekit, at least one therapeutic agent comprises a nucleic acid molecule.In other embodiments of the provided kit, each therapeutic agentcomprises a nucleic acid molecule.

In some embodiments of the provided kit, a first nucleic acid moleculeis a double-stranded oligonucleotide that binds a nucleotide sequenceencoding a TLR2 gene and a second nucleic acid molecule is adouble-stranded oligonucleotide that binds a nucleotide sequenceencoding a TLR4 gene. In some embodiments of the, the double strandedoligonucleotides are linked one to the other in tandem or annealed inRNAistar formation.

In some embodiments of the provided kit, the first double-strandedoligonucleotide comprises:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR24; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand;        and the second double-stranded oligonucleotide comprises:    -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR4; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In various embodiments of the provided kit, the mRNA polynucleotidesequence of TLR2 is set forth in SEQ ID NO:1 and the mRNA polynucleotidesequence of TLR4 is set forth in any one of SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:4.

In some embodiments of the kit, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areformulated for co-administration to a recipient in the same formulation.In other embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are co-administered to thepatient in different formulations. In some embodiments, the firstdouble-stranded oligonucleotide and the second double-strandedoligonucleotide are co-administered to the patient by the same route. Insome embodiments, the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide are co-administered to thepatient by different routes. In various embodiments, the mode ofadministration of the first double-stranded oligonucleotide and thesecond double-stranded oligonucleotide to the recipient of the lungtransplant is selected from the group comprising: separate, combined,simultaneous and sequential administration.

In some embodiments of the provided kit, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areformulated for administering to the patient once. In other embodiments,the first double-stranded oligonucleotide and the second double-strandedoligonucleotide are formulated for administering to the patient at leastonce-a-day. In other embodiments, the first double-strandedoligonucleotide and the second double-stranded oligonucleotide areformulated for multiple administrations to the patient.

In some embodiments of the provided kit, at least one double-strandedoligonucleotide independently comprises a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and

wherein (N)x comprises an antisense sequence to an mRNA selected from anmRNA encoding TLR2 and an mRNA encoding TLR4.

In various embodiments of the provided kit, in structure (A1), the mRNApolynucleotide sequence of TLR2 is set forth in SEQ ID NO:1 and the mRNApolynucleotide sequence of TLR4 is set forth in any one of SEQ ID NO:2,SEQ ID NO:3 and SEQ ID NO:4.

In some preferred embodiments of the provided kit, in structure (A1),x=y=19.

In some embodiments of the provided kit, at least one double-strandedoligonucleotide independently comprises a structure (A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′(sense strand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of the provided kit, in structure (A2), the mRNApolynucleotide sequence of TLR2 is set forth in SEQ ID NO:1 and the mRNApolynucleotide sequence of TLR4 is set forth in any one of SEQ ID NO:2,SEQ ID NO:3 and SEQ ID NO:4.

In some preferred embodiments of the provided kit, in structure (A2),x=y=18.

In another aspect, provided is a package comprising A) at least twoseparate dosage units selected from (i) at least one dosage unitcomprising a TLR2 inhibitor and (ii) at least one dosage unit comprisinga TLR4 inhibitor; and optionally B) a package insert comprisinginstructions for use of the dosage units.

In various embodiments of the provided package, the TLR2 inhibitor is adouble-stranded oligonucleotide that binds a nucleotide sequenceencoding a TLR2 gene and the TLR4 inhibitor is a double-strandedoligonucleotide that binds a nucleotide sequence encoding a TLR4 gene.

In some embodiments of the provided package, the TLR2 inhibitor is adouble-stranded oligonucleotide comprising:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR2; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand;        and the TLR4 inhibitor is a double-stranded oligonucleotide        comprising:    -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR4; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In some embodiments of the provided package, the dosage units areco-administered to a patient by the same route. In other embodiments ofthe package, the dosage units are co-administration to a patient bydifferent routes. In various embodiments, the mode of administration ofthe dosage units is selected from the group comprising: separate,combined, simultaneous and sequential administration.

In some embodiments of the provided package, the dosage units aredesigned for administering to the patient once. In other embodiments,the dosage units are for administering to the patient at leastonce-a-day. In other embodiments, the dosage units are for multipleadministrations to the patient.

In some embodiments of the provided package, at least onedouble-stranded oligonucleotide independently comprises a structure(A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and

wherein (N)x comprises an antisense sequence to an mRNA selected from anmRNA encoding TLR2 and an mRNA encoding TLR4.

In some embodiments of the provided package, at least onedouble-stranded oligonucleotide independently comprises a structure(A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of the provided kit or the provided package, theinstructions or package insert indicates that the therapeutic agents ordosage units are suitable for use in treating a patient suffering from alung disease, injury or disorder selected from the group consisting ofacute respiratory distress syndrome (ARDS), acute lung injury, pulmonaryfibrosis (idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilation induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, a disorder associated with lungtransplantation and emphysema. In some embodiments of the provided kitor the provided package, the instructions or package insert indicatethat the therapeutic agents or dosage units are suitable for use intreating a patient suffering from a disorder associated with lungtransplantation.

In some embodiments of the provided kit or the provided package, thelung disorder associated with lung transplantation is selected from thegroup consisting of inflammation, graft rejection, primary graftfailure, ischemia-reperfusion injury, reperfusion injury, reperfusionedema, allograft dysfunction, acute graft dysfunction, pulmonaryreimplantation response, bronchiolitis obliterans and primary graftdysfunction (PGD).

In another aspect, provided is a method of preventing or reducing thesymptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, comprising administering to the recipient atherapeutically-effective amount of at least one TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof, thereby preventingor reducing the symptoms of PGD in the recipient.

In some embodiments of the provided method, the recipient of a lungtransplant is a human that is being treated for primary graftdysfunction (PGD). In some embodiments, the method is for preventing orreducing the symptoms of cold ischemia-associated PGD. In otherembodiments the method is for preventing or reducing the symptoms ofwarm ischemia-associated PGD. In various embodiments of the providedmethod, the symptoms are selected from the group consisting ofinflammation, acute graft rejection, graft rejection,ischemia-reperfusion injury, reperfusion injury, impaired pulmonaryfunction, bronchiolitis obliterans, impaired blood oxygenation,increased inflammatory cytokine production, intra-graft and intra-airwayaccumulation of granulocytes, pulmonary edema and hypoxemia.

In some embodiments of the provided method, the administration of atherapeutically-effective amount of at least one TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof, results in one ormore of the following: reduced pulmonary edema, increased bloodoxygenation, preserved blood oxygenation, improved pulmonary function,preserved pulmonary function in the recipient of a lung transplant andimproved pulmonary function of the transplanted lung.

In various embodiments of the provided method, the at least one TLR2inhibitor is administered to the recipient of a lung transplant priorto, during or following the lung transplantation.

In various embodiments the provided method of preventing or reducing thesymptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, further comprises at least one additional treatment selectedfrom the group consisting of surgery, steroid therapy, non-steroidtherapy, antiviral therapy, antifungal therapy, antimicrobial therapy,immunosuppressant therapy, anti-infective therapy, anti-hypertensivetherapy, nutritional supplements and any combination thereof. In variousembodiments, the additional treatment is administered prior to,subsequent to or concomitantly with administering of at least one TLR2inhibitor. In some embodiments, the additional treatment comprisesimmunosuppressant therapy.

In various embodiments of the provided method, the route ofadministration of at least one TLR2 inhibitor is selected from: systemicadministration or local administration.

In various embodiments, the method of administration of at least oneTLR2 inhibitor to the recipient of a lung transplant is selected fromthe group comprising: intravenous, intraarterial, intraperitoneal,intramuscular, intraportal, subcutaneous, direct injection,intratracheal instillation, inhalation, intranasal, pulmonary andadministration via pump into the lung. In some embodiments, at least oneTLR2 inhibitor is administered to the recipient of a lung transplant byinhalation. In another embodiments, at least one TLR2 inhibitor isadministered to the recipient of a lung transplant by intratrachealinstillation.

In various embodiments of the provided method of preventing or reducingthe symptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, the at least one TLR2 inhibitor is selected from the groupconsisting of a small organic molecule, a protein, an antibody orfragment thereof, a peptide, a peptidomimetic and a nucleic acidmolecule. In some embodiments, at least one inhibitor comprises anucleic acid molecule. In some embodiments, the nucleic acid molecule isa double-stranded oligonucleotide that binds a nucleotide sequenceencoding a TLR2 gene.

In various embodiments of the provided method of preventing or reducingthe symptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant, the double-stranded oligonucleotide comprises:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR2; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In various embodiments of the provided method, the double-strandedoligonucleotide is formulated for administering to the recipient once.In some embodiments of the provided method, the double-strandedoligonucleotide is formulated for administering to the recipient atleast once-a-day. In yet other embodiments, the double-strandedoligonucleotide is formulated for multiple administrations to therecipient.

In various embodiments of the provided method, the double-strandedoligonucleotide comprises a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y; wherein each of x and yis independently an integer between 17 and 40;wherein the sequence of (N′)y is complementary to the sequence of (N)x;and wherein (N)x comprises an antisense sequence to an mRNA encodingTLR2.

In various embodiments of the provided method, in structure (A1), themRNA polynucleotide sequence of TLR2 is set forth in SEQ ID NO:1.

In some preferred embodiments of the provided method, in structure (A1),x=y=19.

In various embodiments of the provided method, in structure (A1), (N)xcomprises an antisense oligonucleotide present in SEQ ID NOs: 723-1440and 2247-3052 and (N′)y comprises a sense strand oligonucleotide presentin SEQ ID NOs: 5-722 and 1441-2246.

In various embodiments of the provided method, the double-strandedcompound comprises a structure (A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand wherein (N)x is complementary to a consecutive sequence in an mRNAencoding TLR2;

wherein N1 is covalently bound to (N)x and is mismatched to the mRNAencoding TLR2;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of the provided method, in structure (A2), themRNA polynucleotide sequence of TLR2 is set forth in SEQ ID NO:1

In some preferred embodiments of the provided method, in structure (A2),x=y=18.

In various embodiments of the provided method, in structure (A2), thesequence of (N)x is selected from anyone of SEQ ID NOs: 4153-5252 and5546-5838 and the sequence of (N′)y is selected from anyone of SEQ IDNOs: 3053-4152 and 5253-5545.

In various embodiments of the provided method, administration of the atleast one double-stranded oligonucleotide that binds a nucleotidesequence encoding a TLR2 gene results in down-regulation of TLR2expression.

In another aspect provided is a kit or package comprising at least onedosage unit comprising a TLR2 inhibitor; optionally with instructionsfor use, wherein the instructions indicate that the dosage unit issuitable for use in treating a patient suffering from a lung disease,injury or disorder selected from the group consisting of respiratorydistress syndrome (ARDS), acute lung injury, pulmonary fibrosis(idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilator induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, a disorder associated with lungtransplantation and emphysema.

In some embodiments the provided kit or package are for use in treatinga patient suffering from a disorder associated with lungtransplantation.

In various embodiments of the provided kit or package, the TLR2inhibitor is selected from the group consisting of a small organicmolecule, a protein, an antibody or fragment thereof, a peptide, apeptidomimetic and nucleic acid molecule. In some embodiments of the kitor package, the TLR2 inhibitor is selected from the group consisting ofa small organic molecule, a protein; an antibody or fragment thereof;and a nucleic acid molecule. In other embodiments of the kit or package,the TLR2 inhibitor comprises a nucleic acid molecule.

In some embodiments of the provided kit or package, the nucleic acidmolecule is a double-stranded oligonucleotide that binds a nucleotidesequence encoding a TLR2 gene. In some embodiments of the kit orpackage, the double-stranded oligonucleotide comprises:

-   -   (a) a sense strand and an antisense strand;    -   (b) each strand is independently 17 to 40 nucleotides in length;    -   (c) a 17 to 40 nucleotide sequence of the antisense strand is        complementary to a sequence of an mRNA encoding TLR2; and    -   (d) a 17 to 40 nucleotide sequence of the sense strand is        complementary to the antisense strand.

In some embodiments of the provided kit or package, the double-strandedoligonucleotide is formulated for administering to the patient once. Insome embodiments, the double-stranded oligonucleotide is formulated foradministering to the patient at least once-a-day. In some embodiments ofthe provided kit or package, the double-stranded oligonucleotide isformulated for multiple administrations to the patient.

In some embodiments of the provided kit or package, the double-strandedoligonucleotide comprises a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and wherein (N)x comprises an antisense sequence to an mRNA encodingTLR2.

In some embodiments of the provided kit or package, the double-strandedoligonucleotide comprises a structure (A2):

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA encodingTLR2;

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAencoding TLR2;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In some embodiments, the provided kit or package is for use in treatinga patient suffering from a disorder associated with lungtransplantation. In various embodiments the disorder associated withlung transplantation is selected from the group consisting ofinflammation, graft rejection, primary graft failure,ischemia-reperfusion injury, reperfusion injury, reperfusion edema,allograft dysfunction, acute graft dysfunction, pulmonary reimplantationresponse, bronchiolitis obliterans and primary graft dysfunction (PGD).

In another aspect provided is a use of a composition comprising at leastone TLR2 inhibitor or a pharmaceutically acceptable salt or prodrugthereof and at least one TLR4 inhibitor or a pharmaceutically acceptablesalt or prodrug thereof; and a pharmaceutically acceptable carrier, forthe preparation of a medicament for treating or preventing or reducingthe symptoms of primary graft dysfunction (PGD) in a recipient of a lungtransplant.

In another aspect provided is a use of a composition comprising at leastone TLR2 inhibitor or a pharmaceutically acceptable salt or prodrugthereof and at least one TLR4 inhibitor or a pharmaceutically acceptablesalt or prodrug thereof; and a pharmaceutically acceptable carrier, fortreating or preventing or reducing the symptoms of primary graftdysfunction (PGD) in a recipient of a lung transplant.

In various embodiments of the provided use, the recipient of a lungtransplant is a human that is being treated for primary graftdysfunction (PGD). In some embodiments, the use is for preventing orreducing the symptoms of cold ischemia-associated PGD. In otherembodiments, the use is for preventing or reducing the symptoms of warmischemia-associated PGD.

In various embodiments of the use, the symptoms are selected from thegroup consisting of inflammation, acute graft rejection, graftrejection, ischemia-reperfusion injury, reperfusion injury, impairedpulmonary function, bronchiolitis obliterans, impaired bloodoxygenation, increased inflammatory cytokine production, intra-graft andintra-airway accumulation of granulocytes, pulmonary edema andhypoxemia.

In another aspect provided is a use of a composition comprising at leastone TLR2 inhibitor or a pharmaceutically acceptable salt or prodrugthereof and at least one TLR4 inhibitor or a pharmaceutically acceptablesalt or prodrug thereof; and a pharmaceutically acceptable carrier, forthe preparation of a medicament for treating or preventing a lungdisease, disorder or injury selected from acute respiratory distresssyndrome (ARDS), acute lung injury, pulmonary fibrosis (idiopathic),bleomycin induced pulmonary fibrosis, mechanical ventilator induced lunginjury, chronic obstructive pulmonary disease (COPD), chronicbronchitis, and emphysema.

In another aspect provided is a use of a composition comprising at leastone TLR2 inhibitor or a pharmaceutically acceptable salt or prodrugthereof and at least one TLR4 inhibitor or a pharmaceutically acceptablesalt or prodrug thereof; and a pharmaceutically acceptable carrier, fortreating or preventing a lung disease, disorder or injury selected fromacute respiratory distress syndrome (ARDS), acute lung injury, pulmonaryfibrosis (idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilator induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, and emphysema.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that combined administration of a double-stranded RNA(dsRNA) specific for TLR2, at a dose of 25 μg/mouse and adouble-stranded RNA (dsRNA) specific for TLR4, at a dose of 25 μg/mouseefficiently reduced post-transplantation lung edema and hemorrhages inthe transplanted mouse lung. Photographs of the recipient's lung weretaken at 24 hours after orthotopic lung transplantation. Left: dsRNAcombination (combination of dsRNA specific for TLR2 and dsRNA specificfor TLR4, each at 25 μg/mouse (identified in the figure as “siRNAcocktail, 25 μg”)) was administered at the end of lung transplantationsurgery (immediately after anastomosis opening), by intratrachealinstillation to the recipient. Right: vehicle. Arrows: prominenthemorrhages.

FIG. 2 shows that dual target dsRNA combination, targeting TLR2 and TLR4genes z93rd and 4^(th) columns), restored pulmonary function in therecipient's lung. Oxygenation of the arterial blood in mice was measuredat 24 h after lung transplantation and dsRNA administration.Administration of a single dsRNA targeting TLR2 (5^(th) and 6^(th)columns), was also significantly effective in preserving pulmonaryfunction. While administration of a single dsRNA targeting TLR4 (8^(th)and 9^(th) columns), was not effective in preserving pulmonary function.Control groups were composed of (i) mice that were administered withvehicle (general negative control, 2^(nd) column), and (ii) mice thatunderwent lung transplantation (Tx) after only 1 hour of coldpreservation (1 hour cold ischemia time (CIT)) (reperfusion control,1^(st) column).

FIG. 3 shows that dual target dsRNA combination, targeting TLR2 and TLR4genes (columns 4-6), restored pulmonary function in the recipient'slung. Oxygenation of the arterial blood in mice was measured at 24 hafter lung transplantation and dsRNA administration. Negative controlgroups were composed of normal (intact) mice (general negative control,column 1), as well as mice that underwent lung transplantation (Tx)after only 1 hour of cold preservation (1 hour cold ischemia time (CIT))(reperfusion control, column 2) and mice that were treated with avehicle (18 hour cold ischemia time (CIT), column 3).

FIG. 4 shows that a combination of dsRNA specific for TLR2 and dsRNAspecific for TLR4 (TLR2_4_S73 and TLR4_4_S500) (column 3), as well as anindividual treatment comprising dsRNA specific for TLR2 (TLR2_4_S73)(columns 4-6), diminishes intra-airway accumulation of granulocytes inthe BAL obtained from transplanted lungs. At 24 h after lungtransplantation, BAL was collected from all the mice. Total amount ofcells, as well as amounts of different cell populations (neutrophils,lymphocytes, monocytes, eosinophils, basophils) were measured by FACS.Differential cell counts are presented as fractions of total cellcounts.

FIG. 5 shows that treatment with a combination of dsRNA specific forTLR2 and dsRNA specific for TLR4 (TLR2_4_S73 and TLR4_4_S500 (identifiedin the figure as “siRNA cocktail”)) diminished abundance of intragraftIFNγ⁺ CD8⁺ T cells on day 7 post allogeneic transplantation. (A). FACSdemonstrating representative percent abundance of intragraft IFNγ⁺ CD8⁺T cells (N>6); (B) Plotted percent abundance of intragraft IFNγ⁺ CD8⁺ Tcells.

FIG. 6 shows that treatment with a combination of dsRNA specific forTLR2 and dsRNA specific for TLR4 (TLR2_4_S73 and TLR4_4_S500) on days 0and 1, significantly reduced histopathological signs of acute graftrejection in co-stimulation blockade-treated 1 h CIT or 18H CITBalb/c->B6 transplants, treated intratracheally with either controldsRNA (EGFP_5_S763), or a combination of dsRNA specific for TLR2 anddsRNA specific for TLR4 (TLR2_4_S73 and TLR4_4_S500) (identified as“siRNA cocktail”). (A.) Representative histopathological images (HE) ofthe recipient lungs on day 7 post allogeneic lung transplantation. (B).Rejection scores evaluated by board-certified lung transplantpathologist in a blinded fashion. The scoring system is typically usedin the clinic, as follows: Grade A0 (none), Grade A1 (minimal), Grade A2(mild), Grade A3 (moderate) and Grade A4 (severe).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosures relate in part to a method for treating a lungdisorder, disease or injury in a mammal in need thereof. The method mayinclude administering to the mammal at least one therapeutic agentsselected from a TLR2 inhibitor or a pharmaceutically acceptable salt orprodrug thereof; in an amount effective to treat the mammal. The methodmay include administering to the mammal at least two therapeutic agentswherein at least one therapeutic agent targets the TLR2 gene or geneproduct and at least one therapeutic agent targets the TLR4 gene or geneproduct. In some embodiments the therapeutic agents include: (i) a TLR2inhibitor or a pharmaceutically acceptable salt or prodrug thereof, and(ii) a TLR4 inhibitor or a pharmaceutically acceptable salt or prodrugthereof; in amounts effective to treat the lung disorder, disease orinjury in the mammal. The present disclosures also relate tocombinations, compositions, kits and packages that include thetherapeutic agents.

In some embodiments, methods may include administering to the mammal atleast one therapeutic agents in an amount sufficient to reduceexpression and/or to inhibit function of TLR2 gene. In some embodimentsmethods may include administering to the mammal a combination of atleast two therapeutic agents or a combined therapeutic agent in anamount sufficient to reduce expression and/or to inhibit function ofboth a TLR2 gene and a TLR4 gene. In certain embodiments the lungdisease or injury is selected from the group consisting of acuterespiratory distress syndrome (ARDS), acute lung injury, pulmonaryfibrosis (idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilator induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, emphysema, lung transplantation-inducedacute graft dysfunction and bronchiolitis obliterans after lungtransplantation. In certain embodiments, provided are compositions andmethods for combination therapy for treating or preventing inflammationand/or graft rejection associated with organ transplantation, inparticular lung transplantation, including treatment, prevention orattenuation of progression of primary graft failure,ischemia-reperfusion injury, reperfusion injury, reperfusion edema,allograft dysfunction, pulmonary reimplantation response, bronchiolitisobliterans after lung transplantation and/or primary graft dysfunction(PGD) after organ transplantation, in particular in lungtransplantation.

In some embodiments the at least one therapeutic agent is a TLR2inhibitor. In some embodiments the at least two therapeutic agents are aTLR2 inhibitor and a TLR4 inhibitor. In some embodiments the at leasttwo therapeutic agents are co-administered, e.g. concomitantly or insequence. In other embodiments, the at least two therapeutic agents areadministered in a pharmaceutical composition comprising a combinationthereof. In some embodiments the therapeutic agent is a combinedinhibitor by which it is meant a single agent which is capable ofdown-regulating the expression and/or activity of both gene TLR2 andgene TLR4 and/or gene products thereof. Non-limiting examples of suchsingle agents are tandem and multi-armed RNAi molecules disclosed in PCTPatent Publication No. WO 2007/091269.

In one embodiment the method comprises administering a therapeuticallyeffective amount of a therapeutic agent, which targets TLR2.

In some embodiments the method comprises administering (a) atherapeutically effective amount of a first therapeutic agent, whichtargets TLR2 and (b) a therapeutically effective amount of a secondtherapeutic agent, which targets TLR4.

In one embodiment the method comprises administering a therapeuticallyeffective amount of a combined inhibitor, which targets both TLR2 andTLR4.

In some embodiments the therapeutic agent is a TLR2 inhibitor. In someembodiments the therapeutic agent is selected from the group consistingof a small organic molecule chemical compound; a protein; an antibody orfragment thereof; a peptide; a peptidomimetic and a nucleic acidmolecule. In some embodiments at least one therapeutic agent is anucleic acid molecule. In some embodiments the therapeutic agentcomprises a nucleic acid molecule. In some embodiments the nucleic acidmolecule is independently selected from the group consisting of anantisense molecule, a short interfering nucleic acid (siNA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA)or short hairpin RNA (shRNA) that bind a nucleotide sequence (such as anmRNA sequence) encoding the gene TLR2, for example the mRNA codingsequence for human TLR2 exemplified by SEQ ID NO:1(gi|68160956|ref|NM_003264.3|).

In some embodiments the at least two therapeutic agents are a TLR2inhibitor and a TLR4 inhibitor. In some embodiments each therapeuticagent is independently selected from the group consisting of a smallorganic molecule; a protein; an antibody or fragment thereof; a peptide;a peptidomimetic and a nucleic acid molecule. In some embodiments atleast one therapeutic agent is a nucleic acid molecule. In someembodiments each therapeutic agent comprises a nucleic acid molecule.

In some embodiments each nucleic acid molecule is independently selectedfrom the group consisting of an antisense molecule, a short interferingnucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA(dsRNA), micro-RNA (miRNA) or short hairpin RNA (shRNA) that bind anucleotide sequence (such as an mRNA sequence) encoding a target geneselected from TLR2 and TLR4, for example: the mRNA coding sequence forhuman TLR2 exemplified by SEQ ID NO:1 or the mRNA coding sequence forhuman TLR4 exemplified by SEQ ID NOs:2-4. In various embodiments eachnucleic acid molecule is a dsRNA molecule or a siRNA molecule.

In various embodiments each therapeutic agent comprises a nucleic acidmolecule, wherein:

(a) the nucleic acid molecule includes a sense strand and an antisensestrand;

(b) each strand of the nucleic acid molecule is independently 17 to 40nucleotides in length;

(c) a 17 to 40 nucleotide sequence of the antisense strand iscomplementary to a sequence of an mRNA selected from an mRNA encodingTLR2 (e.g., SEQ ID NO: 1) or an mRNA encoding TLR4 (e.g., SEQ ID NOs:2-4); and

(d) a 17 to 40 nucleotide sequence of the sense strand is complementaryto the antisense strand and includes a 17 to 40 nucleotide sequence of amRNA selected from a mRNA encoding TLR2 (e.g., SEQ ID NO: 1) and an mRNAencoding TLR4 (e.g., SEQ ID NOs: 2-4).

In various embodiments each therapeutic agent comprises a nucleic acidmolecule having a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and

wherein (N)x comprises an antisense sequence to an mRNA selected from anmRNA encoding TLR2 and an mRNA encoding TLR4.

In some embodiments the sequence of TLR2 mRNA is set forth in SEQ IDNO:1. In various embodiments the sense and antisense strands of the TLR2siRNA oligonucleotides are selected from the sense strand sequences setforth in SEQ ID NOs: 5-722; 1441-2246; 3053-4152; and 5253-5545 andantisense strand sequences set forth in SEQ ID NOs: 723-1440; 2247-3052;4153-5252 and 5546-5838. In some embodiments the sequence of TLR4 mRNAis set forth in SEQ ID NO:2; SEQ ID NO:3 or SEQ ID NO:4. In variousembodiments the sense and antisense strands of the TLR4 siRNAoligonucleotides are selected from the sense strand sequences set forthin SEQ ID NOs: 5839-7075, 8313-8458, 8605-10318, 12033-12084 andantisense strand sequences set forth in SEQ ID NOs: 7076-8312,8459-8604, 10319-12032, 12085-12136.

In some embodiments (N)x of the double-stranded oligonucleotide compoundcomprises an antisense oligonucleotide present in SEQ ID NOs: 723-1440,2247-3052, 4153-5252, 5546-5838, 7076-8312, 8459-8604, 10319-12032,12085-12136. In some embodiments the sequence of (N′)y is partiallycomplementary to the sequence of (N)x. In some embodiments the sequenceof (N′)y is substantially complementary to the sequence of (N)x. In someembodiments the sequence of (N′)y is fully complementary to the sequenceof (N)x. In some embodiments (N)x of the double-stranded oligonucleotidecompound comprises an antisense oligonucleotide present indouble-stranded RNA compounds identified as TLR2_4, TLR2_7 or TLR4_4.

In some embodiments of the double-stranded oligonucleotide compoundx=y=19. In various embodiments both Z and Z′ are present in thedouble-stranded oligonucleotide compound. In various embodiments both Zand Z′ are absent in the double-stranded oligonucleotide compound; i.e.the double-stranded compound is blunt ended on both ends. In someembodiments at least one of Z or Z′ is present in said double-strandedoligonucleotide compound.

In some embodiments Z or Z′ is independently an unconventional moietyselected from an abasic deoxyribose moiety, an abasic ribose moiety aninverted abasic deoxyribose moiety, an inverted abasic ribose moiety; aC3 moiety, a C4 moiety, a C5 moiety, an amino-6 moiety. In somepreferred embodiments Z or Z′ is independently selected from a C3 moietyand an amino-C6 moiety.

In some embodiments at least one of N or N′ in the double-strandedoligonucleotide compound comprises a 2′ sugar modified ribonucleotide.In some embodiments the 2′ sugar modification comprises the presence ofan amino, a fluoro, an alkoxy or an alkyl moiety. In some preferredembodiments 2′ sugar modification comprises the presence of an alkoxymoiety, preferably the alkoxy moiety comprises a 2′-O-Methyl moiety.

In some embodiments of the double-stranded oligonucleotide compound,(N)x comprises alternating 2′-O-Methyl sugar modified ribonucleotidesand unmodified ribonucleotides. In certain embodiments, (N)x comprisesat least 5 alternating 2′-O-Methyl sugar modified and unmodifiedribonucleotides. In some embodiments, (N)x comprises 2′-O-Methyl sugarmodified ribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19.In some embodiments, (N)x comprises 2′-O-Methyl sugar modifiedribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. Insome embodiments, (N)x comprises 2′-O-Methyl sugar modified pyrimidineribonucleotides. In some embodiments, all pyrimidine ribonucleotides in(N)x comprise 2′-O-Methyl sugar modified pyrimidine ribonucleotides.

In some embodiments, (N)x comprises at least one unconventional moietyselected from a mirror nucleotide and a nucleotide joined to an adjacentnucleotide by a 2′-5′ internucleotide phosphate bond. In someembodiments, the unconventional moiety in (N)x is a mirror nucleotide.In some embodiments, the mirror nucleotide in (N)x is anL-deoxyribonucleotide (L-DNA). In various embodiments, (N)x comprises anL-DNA moiety at position 6 or 7 (5′>3′).

In some embodiments, (N′)y comprises at least one unconventional moietyselected from a mirror nucleotide and a nucleotide joined to an adjacentnucleotide by a 2′-5′ internucleotide phosphate bond. In someembodiments, the unconventional moiety in (N′)y is a mirror nucleotide.In some embodiments, the mirror nucleotide in (N′)y is anL-deoxyribonucleotide (L-DNA). In some embodiments, (N′)y consists ofunmodified ribonucleotides at positions 1-17 and 19 and one L-DNA at the3′ penultimate position (position 18). In some embodiments, (N′)yconsists of unmodified ribonucleotides at position 1-16 and 19 and twoconsecutive L-DNA at the 3′ penultimate positions (positions 17 and 18).In some embodiments the unconventional moiety in (N′)y is a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide phosphatelinkage. In some embodiments, in (N′)y the nucleotide joined to anadjacent nucleotide by a 2′-5′ internucleotide phosphate linkage furthercomprises a 3′-O-Methyl (3′O-Me) sugar modification.

In various embodiments the therapeutic agent is a double-strandedoligonucleotide compound having a structure (A2) set forth below:

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA selectedfrom an mRNA encoding TLR2 (e.g., SEQ ID NO: 1) and an mRNA encodingTLR4 (e.g., SEQ ID NOs: 2-4);

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAselected from an mRNA encoding TLR2 (e.g., SEQ ID NO: 1) and an mRNAencoding TLR4 (e.g., SEQ ID NOs: 2-4);

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In some embodiments of the double-stranded oligonucleotide compoundaccording to Structure (A)2, x=y=18.

In some embodiments (N)x is complementary to a consecutive sequence inSEQ ID NO:1 (human TLR2 mRNA). In some embodiments (N)x includes anantisense oligonucleotide selected from any one of SEQ ID NOs: 4153-5252and 5546-5838. In some embodiments x=y=18 and N1-(N)x includes anantisense oligonucleotide selected from any one of SEQ ID NOs: 723-1440and 2247-3052. In some embodiments x=y=19 or x=y=20. In certainpreferred embodiments x=y=18.

In some embodiments (N)x is complementary to a consecutive sequence inSEQ ID NO:2 (human TLR4, transcript variant 4, non-coding RNA) or SEQ IDNO:3 (human TLR4, transcript variant 1, mRNA) or SEQ ID NO:4 (humanTLR4, transcript variant 3, non-coding RNA). In some embodiments (N)xincludes an antisense oligonucleotide selected from any one of SEQ IDNOs: 10319-12032 and 12085-12136. In some embodiments x=y=18 and N1-(N)xincludes an antisense oligonucleotide selected from any one of SEQ IDNOs: 7076-8312 and 8459-8604. In some embodiments x=y=19 or x=y=20. Incertain preferred embodiments x=y=18.

In some embodiments N1 and N2 form a Watson-Crick base pair. In otherembodiments N1 and N2 form a non-Watson-Crick base pair. In someembodiments N1 is a modified riboadenosine or a modified ribouridine.

In certain embodiments N1 is selected from the group consisting ofriboadenosine, modified riboadenosine, deoxyriboadenosine, modifieddeoxyriboadenosine. In other embodiments N1 is selected from the groupconsisting of ribouridine, deoxyribouridine, modified ribouridine, andmodified deoxyribouridine.

In certain embodiments, N1 is selected from the group consisting ofriboadenosine, modified riboadenosine, deoxyriboadenosine, modifieddeoxyriboadenosine and N2 is selected from the group consisting ofribouridine, deoxyribouridine, modified ribouridine, and modifieddeoxyribouridine. In certain embodiments N1 is selected from the groupconsisting of riboadenosine and modified riboadenosine and N2 isselected from the group consisting of ribouridine and modifiedribouridine.

In certain embodiments, N2 is selected from the group consisting ofriboadenosine, modified riboadenosine, deoxyriboadenosine, modifieddeoxyriboadenosine and N1 is selected from the group consisting ofribouridinc, deoxyribouridine, modified ribouridine, and modifieddeoxyribouridine. In certain embodiments, N1 is selected from the groupconsisting of ribouridine and modified ribouridine and N2 is selectedfrom the group consisting of riboadenine and modified riboadenine. Incertain embodiments, N1 is ribouridine and N2 is riboadenine.

In some embodiments of (A2), (N)x is selected from any one of SEQ IDNOs: 4153-5252 and 5546-5838 and (N′)y is a substantially complementarysequence selected from SEQ ID NOs: 3053-4152 and 5253-5545. In someembodiments of (A2), (N)x is selected from any one of SEQ ID NOs:10319-12032 and 12085-12136 and (N′)y is a substantially complementarysequence selected from SEQ ID NOs: 8605-10318 and 12033-12084. In someembodiments the sequence of (N′)y is partially complementary to thesequence of (N)x. In some embodiments the sequence of (N′)y is fullycomplementary to the sequence of (N)x. In some embodiments, (N)x of thedouble-stranded oligonucleotide compound comprises an antisenseoligonucleotide present in double-stranded RNA compounds identified asTLR2_4, TLR2_7 or TLR4_4.

In some embodiments, the administration method is systemicadministration. In some embodiments, the administration method is localadministration. In various embodiments the administration method isintratracheal, inhalant, intravenous, intraarterial, intraperitoneal,intramuscular, intraportal, subcutaneous, intradermal, topical, directadministration into a target lung tissue by injection or via a pump.

In one aspect provided is a pharmaceutical composition that includes atleast one therapeutic agent selected from a TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof; and apharmaceutically acceptable carrier.

In another aspect provided is a combination that includes at least twotherapeutic agents selected from: (i) a TLR2 inhibitor or apharmaceutically acceptable salt or prodrug thereof and (ii) a TLR4inhibitor or a pharmaceutically acceptable salt or prodrug thereof; anda pharmaceutically acceptable carrier.

In another aspect provided is a pharmaceutical composition that includesa combination of at least two therapeutic agents selected from: (i) aTLR2 inhibitor or a pharmaceutically acceptable salt or prodrug thereofand (iii) a TLR4 inhibitor or a pharmaceutically acceptable salt orprodrug thereof; and a pharmaceutically acceptable carrier.

In some embodiments the composition comprises a therapeutic agentconsisting of a TLR2 inhibitor. In some embodiments the combination orcomposition comprises at least two therapeutic agents, wherein at leastone of the therapeutic agents is a TLR2 inhibitor or a pharmaceuticallyacceptable salt or prodrug thereof, and at least one of the therapeuticagents is a TLR4 inhibitor or a pharmaceutically acceptable salt orprodrug thereof. In some embodiments the combination or compositioncomprises a TLR2 inhibitor and a TLR4 inhibitor.

In some embodiments the TLR2 inhibitor is selected from the groupconsisting of a small molecule chemical compound; a protein; an antibodyor fragment thereof; and a nucleic acid molecule. In some embodimentsthe TLR2 inhibitor comprises a nucleic acid molecule. In someembodiments the nucleic acid molecule is selected from a shortinterfering nucleic acid (siNA), a short interfering RNA (siRNA), adouble-stranded RNA (dsRNA), a micro-RNA (miRNA) or short hairpin RNA(shRNA) that binds a nucleotide sequence (such as an mRNA sequence)encoding the target gene TLR2. In some embodiments the nucleic acidmolecule is a double-stranded RNA (dsRNA) or a short interfering RNA(siRNA) targeting TLR2.

In some embodiments each therapeutic agent is independently selectedfrom the group consisting of a small molecule chemical compound; aprotein; an antibody or fragment thereof; and a nucleic acid molecule.In some embodiments each therapeutic agent comprises a nucleic acidmolecule. In some embodiments each nucleic acid molecule isindependently selected from a short interfering nucleic acid (siNA), ashort interfering RNA (siRNA), a double-stranded RNA (dsRNA), amicro-RNA (miRNA) or short hairpin RNA (shRNA) that binds a nucleotidesequence (such as an mRNA sequence) encoding the target gene selectedfrom TLR2 and TLR4. In some embodiments each nucleic acid molecule is adouble-stranded RNA (dsRNA) or a short interfering RNA (siRNA). In someembodiments the at least two dsRNA or siRNA are a dsRNA or siRNAtargeting TLR2 and a dsRNA or siRNA targeting TLR4.

In one embodiment the method comprises a therapeutically effectiveamount of a therapeutic agent, which down-regulates TLR2.

In one embodiment the method comprises (a) a therapeutically effectiveamount of a first therapeutic agent, which down-regulates TLR2 and (b) atherapeutically effective amount of a second therapeutic agent, whichdown-regulates TLR4.

In another aspect provided is a kit comprising at a therapeutic agentconsisting of a TLR2 inhibitor; optionally with instructions for use.

In another aspect provided is a kit comprising at least two therapeuticagents wherein the two agents are selected from the group consisting ofa TLR2 inhibitor and a TLR4 inhibitor; optionally with instructions foruse.

In some embodiments of the kit each therapeutic agent is independentlyselected from the group consisting of a small molecule chemicalcompound; a protein; an antibody or fragment thereof; and a nucleic acidmolecule. In some embodiments each therapeutic agent comprises a nucleicacid molecule. In some embodiments each nucleic acid molecule isindependently selected from a short interfering nucleic acid (siNA), ashort interfering RNA (siRNA), a double-stranded RNA (dsRNA), amicro-RNA (miRNA) or short hairpin RNA (shRNA) that binds a nucleotidesequence (such as an mRNA sequence) encoding a target gene selected fromTLR2 and TLR4. In some embodiments each nucleic acid molecule is adouble-stranded RNA (dsRNA) or a short interfering RNA (siRNA). In someembodiments each nucleic acid molecule is selected from the groupconsisting of a dsRNA targeting TLR2 or a siRNA targeting TLR2; and adsRNA targeting TLR4 or a siRNA targeting TLR4. In some embodiments theat least two siRNA consist of: a dsRNA or siRNA targeting TLR2; and adsRNA or siRNA targeting TLR4.

In some embodiments a kit provided herein comprises a combined inhibitorby which it is meant a single agent which is capable of down-regulatingat least two genes and/or gene products selected from the groupconsisting both TLR2 and TLR4; optionally with instructions for use.

In some embodiments each therapeutic agent of the kit comprises anucleic acid molecule, wherein:

(a) the nucleic acid molecule includes a sense strand and an antisensestrand;

(b) each strand of the nucleic acid molecule is independently 17 to 40nucleotides in length;

(c) a 17 to 40 nucleotide sequence of the antisense strand iscomplementary to a sequence of an mRNA selected from an mRNA encodingTLR2 (e.g., SEQ ID NO: 1) and an mRNA encoding TLR4 (e.g., SEQ ID NOs:2-4); and

(d) a 17 to 40 nucleotide sequence of the sense strand is complementaryto the antisense strand and includes a 17 to 40 nucleotide sequence of amRNA selected from a mRNA encoding TLR2 (e.g., SEQ ID NO: 1) and an mRNAencoding TLR4 (e.g., SEQ ID NOs: 2-4).

In some embodiments each therapeutic agent of the kit comprises anucleic acid molecule having a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and wherein (N)x comprises an antisense sequence to an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4.

In various embodiments the double-stranded molecule comprises a mismatchto the target mRNA at the 5′ terminal nucleotide of the guide strand(antisense strand). Accordingly, in some embodiments each therapeuticagent of the kit comprises a double-stranded oligonucleotide compoundhaving a structure (A2) set forth below

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA selectedfrom an mRNA encoding TLR2 (e.g., SEQ ID NO: 1) and an mRNA encodingTLR4 (e.g., SEQ ID NOs: 2-4);

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAselected from an mRNA encoding TLR2 (e.g., SEQ ID NO: 1) and an mRNAencoding TLR4 (e.g., SEQ ID NOs: 2-4);

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In another aspect provided is a package comprising A) at least twoseparate dosage units selected from (i) a dosage unit comprising a TLR2inhibitor, and (ii) a dosage unit comprising a TLR4 inhibitor; andoptionally B) a package insert comprising instructions for use of thedosage units.

In another embodiment of the package each inhibitor comprises a nucleicacid molecule, wherein:

(a) the nucleic acid molecule includes a sense strand and an antisensestrand;

(b) each strand of the nucleic acid molecule is independently 17 to 40nucleotides in length;

(c) a 17 to 40 nucleotide sequence of the antisense strand iscomplementary to a sequence of an mRNA selected from an mRNA encodingTLR2 and an mRNA encoding TLR4; and

(d) a 17 to 40 nucleotide sequence of the sense strand is complementaryto the antisense strand and includes a 17 to 40 nucleotide sequence of amRNA selected from an mRNA encoding TLR2 and an mRNA encoding TLR4.

In some embodiments of the package each inhibitor comprises a nucleicacid molecule having a structure (A1):

(A1) 5′ (N)x-Z 3′ (antisense strand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is a ribonucleotide which may be unmodified ormodified, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of (N′)y;

wherein each of x and y is independently an integer between 17 and 40;

wherein the sequence of (N′)y is complementary to the sequence of (N)x;and wherein (N)x comprises an antisense sequence to an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4.

In various embodiments the double-stranded molecule comprises a mismatchto the target mRNA at the 5′ terminal nucleotide of the guide strand(antisense strand). Accordingly, in some embodiments of the package eachinhibitor comprises a double-stranded oligonucleotide compound having astructure (A2) set forth below:

(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;

wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′)y is complementary to the sequence of (N)xand (N)x is complementary to a consecutive sequence in an mRNA selectedfrom an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is covalently bound to (N)x and is mismatched to an mRNAselected from an mRNA encoding TLR2 and an mRNA encoding TLR4;

wherein N1 is a moiety selected from the group consisting of uridine,modified uridine, ribothymidine, modified ribothymidine,deoxyribothymidine, modified deoxyribothymidine, riboadenine,deoxyriboadenine and modified deoxyriboadenine,

wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N2-(N′)y; and

wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or unconventionalmoieties or a combination thereof covalently attached at the 3′ terminusof the strand in which it is present.

In various embodiments of the kit or package the instructions or packageinsert indicates that the therapeutic agent or dosage unit or thetherapeutic agents or dosage units are suitable for use in treating apatient suffering from a disease or condition selected from the groupconsisting of acute respiratory distress syndrome (ARDS), acute lunginjury, pulmonary fibrosis (idiopathic), bleomycin induced pulmonaryfibrosis, mechanical ventilator induced lung injury, chronic obstructivepulmonary disease (COPD), chronic bronchitis, emphysema, lungtransplantation-induced acute graft dysfunction and bronchiolitisobliterans after lung transplantation. In various embodiments of the kitor package the instructions or package insert indicates that thetherapeutic agents or dosage units are suitable for use in treating apatient suffering from or at risk of suffering from inflammation and/orgraft rejection associated with organ transplantation, in particularlung transplantation, including, without being limited to, primary graftfailure, ischemia-reperfusion injury, reperfusion injury, reperfusionedema, allograft dysfunction, pulmonary reimplantation response,bronchiolitis obliterans after lung transplantation and/or primary graftdysfunction (PGD) after organ transplantation, in particular in lungtransplantation.

In various embodiments the composition comprises one or moredouble-stranded nucleic acid (dsNA) agents which down-regulate orinhibit the expression/activity/function of a TLR2 gene and/or TLR2 geneproduct including DNA and mRNA.

In various embodiments the combination comprises one or moredouble-stranded nucleic acid (dsNA) agents which down-regulate orinhibit the expression/activity/function of at least two genes and/orgene products including DNA and mRNA selected from: (i) TLR2 and (ii)TLR4.

The mRNA coding sequence for human TLR2 is exemplified by SEQ ID NO:1and the mRNA coding sequence for human TLR4 is exemplified by SEQ IDNO:2, SEQ ID NO:3 and SEQ ID NO:4.

In one embodiment the composition comprises at least one dsNA moleculewhich down-regulates TLR2.

In another embodiment the combination comprises one or more dsNA agentswhich down-regulate TLR2 and TLR4. In one embodiment the combinationcomprises at least one dsNA molecule which down-regulates TLR2 and atleast one dsNA molecule which down-regulates TLR4.

In some embodiments provided is a tandem dsRNA comprising dsRNAtargeting at least both TLR2 and TLR4.

In some embodiments provided is a triple armed structure, also known asRNAistar. Said triple-stranded oligonucleotide comprises anoligoribonucleotide having the general structure:

5′ oligo1 (sense) LINKER A oligo2 (sense) 3′ 3′ oligo1 (antisense)LINKER B oligo3 (sense) 5′ 3′ oligo3 (antisense) LINKER C oligo2(antisense) 5′ or 5′ oligo1 (sense) LINKER A oligo2 (antisense) 3′3′ oligo1 (antisense) LINKER B oligo3 (sense) 5′ 3′ oligo3 (antisense)LINKER C oligo2 (sense) 5′ or 5′ oligo1 (sense) LINKER A oligo3(antisense) 3′ 3′ oligo1 (antisense) LINKER B oligo2 (sense) 5′5′ oligo3 (sense) LINKER C oligo2 (antisense) 3′

Wherein one or more of linker A, linker B or linker C is present; anycombination of two or more oligonucleotides and one or more of linkersA-C is possible, so long as the polarity of the strands and the generalstructure of the molecule remains. Further, if two or more of linkersA-C are present, they may be identical or different. In some embodimentsa “gapped” RNAistar compound is preferred wherein the compound comprisesthree RNA duplexes.

A compound consisting of four ribonucleotide strands forming three RNAduplexes having the general structure:

wherein each of oligo A, oligo B, oligo C, oligo D, oligo E and oligo Frepresents at least 19 consecutive ribonucleotides, wherein from 18 to40 of such consecutive ribonucleotides, in each of oligo A, B, C, D, Eand F comprise a strand of a RNA duplex, wherein each ribonucleotide maybe modified or unmodified′

wherein strand 1 comprises oligo A which is either a sense portion or anantisense portion of a first RNA duplex of the compound, strand 2comprises oligo B which is complementary to at least 19 nucleotides inoligo A, and oligo A and oligo B together form a first RNA duplex thattargets a first target mRNA;

wherein strand 1 further comprises oligo C which is either a senseportion or an antisense strand portion of a second RNA duplex of thecompound, strand 3 comprises oligo D which is complementary to at least19 nucleotides in oligo C and oligo C and oligo D together form a secondRNA duplex that targets a second target mRNA;

wherein strand 4 comprises oligo E which is either a sense portion or anantisense strand portion of a third RNA duplex of the compound, strand 2further comprises oligo F which is complementary to at least 19nucleotides in oligo E and oligo E and oligo F together form a third RNAduplex that targets a third target mRNA; and

wherein linker A is a moiety that covalently links oligo A and oligo C;linker B is a moiety that covalently links oligo B and oligo F, andlinker A and linker B can be the same or different.

In some embodiments the first, second and third RNA duplex target thesame gene, i.e. TLR2. In other embodiments two of the first, second orthird siRNA duplexes target the same mRNA, e.g. TLR2 and the third RNAduplex targets a different mRNA, for example TLR4. In other embodimentstwo of the first, second or third siRNA duplexes target the same mRNA,e.g. TLR4 and the third RNA duplex targets a different mRNA, for exampleTLR2.

“Toll-like receptor 2” or “fir-2” or “TLR-2” or “tlr2” or “TLR2” areused interchangeably and refer to any Toll-like receptor 2 peptide orpolypeptide having any TLR2 protein activity. TLR2 has also beendesignated as CD282 (cluster of differentiation 282). Toll-like receptor2 (or more particularly human TLR2) may have an amino acid sequence thatis the same, or substantially the same, as SEQ ID NO. 1.

“Toll-like receptor 4” or “fir-4” or “TLR-4” or “tlr4” or “TLR4” areused interchangeably and refer to any Toll-like receptor 4 peptide orpolypeptide having any TLR4 protein activity. TLR4 has also beendesignated as CD284 (cluster of differentiation 284). Toll-like receptor4 (or more particularly human TLR4) may have an amino acid sequence thatis the same, or substantially the same, as SEQ ID NO. 2-4.

As used herein the term “nucleotide sequence encoding TLR2 and TLR4”means a nucleotide sequence that codes for a TLR2 and TLR4 protein,respectively, or portion thereof. The term “nucleotide sequence encodingTLR2 and TLR4” is also meant to include TLR2 and TLR4 coding sequencessuch as TLR2 and TLR4 isoforms, mutant TLR2 and TLR4 genes, splicevariants of TLR2 and TLR4 genes, and TLR2 and TLR4 gene polymorphisms. Anucleic acid sequence encoding TLR2 and TLR4 includes mRNA sequencesencoding TLR2 and TLR4, which can also be referred to as TLR2 mRNA andTLR4 mRNA. Exemplary sequence of human TLR2 is SEQ ID NO:1. Exemplarysequences of human TLR4 mRNA are SEQ ID NO:2, SEQ ID NO:3 and SEQ IDNO:4.

In some embodiments the inhibitors or therapeutic agents disclosedherein comprise a molecule, a compound which can down-regulate orinhibit expression and/or function of a gene and/or gene productselected from TLR2 and TLR4. Preferably the therapeutic agent isindependently selected from the group consisting of a small organicmolecule; a protein; an antibody or fragment thereof a peptide, apeptidomimetic and a nucleic acid molecule.

Examples of an antibody includes polyclonal, monoclonal, chimeric,humanized or human antibodies and antigen-binding fragments thereof.Examples of TLR2 binding antibodies are anti-human TLR2 antibody, mousemonoclonal anti-human TLR2, rabbit anti-human TLR2, goat anti-human TLR2and the like which are raised against TLR2.

Examples of an antibody includes polyclonal, monoclonal, chimeric,humanized or human antibodies and antigen-binding fragments thereof.Examples of TLR4 binding antibodies are anti-human TLR4 antibody, mousemonoclonal anti-human TLR4, rabbit anti-human TLR4, goat anti-human TLR4and the like which are raised against TLR4.

In some embodiments the inhibitor or therapeutic agent of the presentdisclosure comprise a peptide. The term “peptide”, as used herein,refers to a compound consisting of from about two to about ninety aminoacid residues wherein the amino group of one amino acid is linked to thecarboxyl group of another amino acid by a peptide bond. Preferredpeptide sequences are short (e.g. 3 to 20 amino acids in length) andlipophilic, such that they can cross cell membranes to a sufficientextent. A peptide can be, for example, derived or removed from a nativeprotein by enzymatic or chemical cleavage, or can be prepared usingconventional peptide synthesis techniques (e.g., solid phase synthesis)or molecular biology techniques (see Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)). A “peptide” can comprise any suitable L- and/orD-amino acid, for example, common o-amino acids (e.g., alanine, glycine,valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid,6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g.,citrulline, homocitrulline, homoserine, norleucine, norvaline,ornithine). The amino, carboxyl and/or other functional groups on apeptide can be free (e.g., unmodified) or protected with a suitableprotecting group. Suitable protecting groups for amino and carboxylgroups, and means for adding or removing protecting groups are known inthe art and are disclosed in, for example, Green and Wuts, “ProtectingGroups in Organic Synthesis”, John Wiley and Sons, 1991. The functionalgroups of a peptide can also be derivatized (e.g., alkylated) usingart-known methods.

In some embodiments the inhibitors or therapeutic agents provided hereininclude a peptidomimetic. The term “peptidomimetic”, as used herein,refers to molecules which are not polypeptides, but which mimic aspectsof their structures and have the same functional groups as peptides,which can inhibit TLR2 or TLR4. Peptidomimetics are designed, forexample, by identifying a peptide inhibitor of TLR2 or TLR4 andmodifying it using amino acid substitutes that advantageously modify theproperties of the peptide, for example by increasing stability and oractivity.

In some embodiments the inhibitors or therapeutic agents disclosedherein include nucleic acid molecules. As used herein, the term “nucleicacid molecule” or “nucleic acid” are used interchangeably and refer toan oligonucleotide, nucleotide or polynucleotide. Variations of “nucleicacid molecule” are described in more detail herein. A nucleic acidmolecule encompasses both modified nucleic acid molecules and unmodifiednucleic acid molecules as described herein. A nucleic acid molecule mayinclude deoxyribonucleotides, ribonucleotides, modified nucleotides ornucleotide analogs in any combination.

As used herein, the term “nucleotide” refers to a chemical moiety havinga sugar (or an analog thereof, or a modified sugar), a nucleotide base(or an analog thereof, or a modified base), and a phosphate group (oranalog thereof, or a modified phosphate group). A nucleotide encompassesboth modified nucleotides or unmodified nucleotides as described herein.As used herein, nucleotides may include deoxyribonucleotides (e.g.,unmodified deoxyribonucleotides), ribonucleotides (e.g., unmodifiedribonucleotides), and modified nucleotide analogs including, inter alia,locked nucleic acids and unlocked nucleic acids, peptide nucleic acids,L-nucleotides (also referred to as mirror nucleotides), ethylene-bridgednucleic acid (ENA), arabinoside, PACE, nucleotides with a 6 carbonsugar, as well as nucleotide analogs (including abasic nucleotides)often considered to be non-nucleotides. In some embodiments, nucleotidesmay be modified in the sugar, nucleotide base and/or in the phosphategroup with any modification known in the art and/or any modificationsuch as modifications described herein. A “polynucleotide” or“oligonucleotide” as used herein refer to a chain of linked nucleotides;polynucleotides and oligonucleotides may likewise have modifications inthe nucleotide sugar, nucleotide bases and phosphate backbones as arewell known in the art and/or are disclosed herein.

As used herein, the term “short interfering nucleic acid”, “siNA”, or“short interfering nucleic acid molecule” refers to any nucleic acidmolecule capable of modulating gene expression or viral replication.Preferably siNA inhibits or down regulates gene expression or viralreplication. siNA includes without limitation nucleic acid moleculesthat are capable of mediating sequence specific RNA interference (RNAi),for example short interfering RNA (siRNA), double-stranded NA (dsNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA(shRNA), short interfering oligonucleotide, short interfering nucleicacid, short interfering modified oligonucleotide, chemically-modifiedsiRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Asused herein, “short interfering nucleic acid”, “siNA”, or “shortinterfering nucleic acid molecule” has the meaning described in moredetail elsewhere herein.

As used herein, the term “complementary” means that a nucleic acid canform hydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. In reference tothe nucleic molecules disclosed herein, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Fully complementary”means that all the contiguous residues of a nucleic acid sequence willform hydrogen bond with the same number of contiguous residues in asecond nucleic acid sequence. In one embodiment, a nucleic acid moleculedisclosed herein includes about 15 to about 35 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 or more) nucleotides that are complementary to one or moretarget nucleic acid molecules or a portion thereof.

As used herein, the term “sense region” refers to a nucleotide sequenceof a dsNA molecule complementary (partially or fully) to an antisenseregion of the dsNA molecule. The sense strand of a dsNA molecule caninclude a nucleic acid sequence having homology with a target nucleicacid sequence. As used herein, “sense strand” refers to nucleic acidmolecule that includes a sense region and may also include additionalnucleotides. The sense strand may be between 17 and 40 nucleotides inlength, for instance, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.

As used herein, the term “antisense region” refers to a nucleotidesequence of a dsNA molecule complementary (partially or fully) to atarget nucleic acid sequence, preferably a target mRNA. The antisensestrand of a dsNA molecule can optionally include a nucleic acid sequencecomplementary to a sense region of the dsNA molecule. As used herein,“antisense strand” refers to nucleic acid molecule that includes anantisense region and may also include additional nucleotides. Theantisense strand may be between 17 and 40 nucleotides in length, forinstance, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.

As used herein, the term “substantially complementary” means theantisense strand includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotidesthat are not complementary to a nucleotide sequence of anoligonucleotide, such as a sense strand or a target mRNA. In someembodiments, an antisense strand may include 1, 2, or 3 nucleotides thatare unpaired, i.e., do not have a corresponding complementary nucleotidein the sense strand or in a target mRNA.

As used herein, the term “RNA” refers to a molecule that includes atleast one ribonucleotide residue.

As used herein, the term “duplex region” refers to the region in twocomplementary or substantially complementary oligonucleotides that formbase pairs with one another, either by Watson-Crick base pairing or anyother manner that allows for a duplex between oligonucleotide strandsthat are complementary or substantially complementary. For example, anoligonucleotide strand having 21 nucleotide units can base pair withanother oligonucleotide of 21 nucleotide units, yet only 19 bases oneach strand are complementary or substantially complementary, such thatthe “duplex region” consists of 19 base pairs. The remaining base pairsmay, for example, exist as 5′ and 3′ overhangs. Further, within theduplex region, 100% complementarity is not required; substantialcomplementarity is allowable within a duplex region. Substantialcomplementarity refers to complementarity between the strands such thatthey are capable of annealing under biological conditions. Techniques toempirically determine if two strands are capable of annealing underbiological conditions are well know in the art. Alternatively, twostrands can be synthesized and added together under biologicalconditions to determine if they anneal to one another.

As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which includes a non-base pairing moiety including butnot limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes withoutlimitation a halogen, alcohol, amine, carboxylic, ester, amide,aldehyde, ketone, ether groups.

An “abasic nucleotide” or “abasic nucleotide analog” as used herein mayalso be often referred to herein and in the art as a pseudo-nucleotideor an unconventional moiety. While a nucleotide is a monomeric unit ofnucleic acid, generally consisting of a ribose or deoxyribose sugar, aphosphate, and a base (adenine, guanine, thymine, or cytosine in DNA;adenine, guanine, uracil, or cytosine in RNA). an abasic orpseudo-nucleotide lacks a base, and thus is not strictly a nucleotide asthe term is generally used in the art. Abasic deoxyribose moietiesinclude for example, abasic deoxyribose-3′-phosphate;1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribosemoieties include inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic5′-phosphate.

The term “capping moiety” (or “z” ″) as used herein includes a moietywhich can be covalently linked to the 5′ terminus of the sense strand((N′)y) and includes abasic ribose moiety, abasic deoxyribose moiety,modifications to abasic ribose and abasic deoxyribose moieties including2′ O alkyl modifications; inverted abasic ribose and abasic deoxyribosemoieties and modifications thereof; C6-imino-Pi; a mirror nucleotideincluding L-DNA and L-RNA; 5′OMe nucleotide; and nucleotide analogsincluding 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

Certain capping moieties may be abasic ribose or abasic deoxyribosemoieties; inverted abasic ribose or inverted abasic deoxyribosemoieties; C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA.The nucleic acid molecules as disclosed herein may be synthesized usingone or more inverted nucleotides, for example inverted thymidine orinverted adenine (for example see Takei, et al., 2002. JBC277(26):23800-06).

In some embodiments of Structure (A1) and Structure (A2) at least one ofZ or Z′ is present and comprises at least two non-nucleotide moietiescovalently attached to the strand in which it is present. In someembodiments each of Z and Z′ independently includes a C3 alkyl, C3alcohol or C3 ester moiety. In some embodiments Z′ is absent and Z ispresent and includes a non-nucleotide C3 moiety. In some embodiments Zis absent and Z′ is present and includes a non-nucleotide C3 moiety.Exemplary non-nucleotide moieties include the alkyl and modified alkylmoieties shown below:

In some embodiments of Structures (A1) and (A2), each of N and N′ is anunmodified nucleotide. In some embodiments at least one of N or N′includes a chemically modified nucleotide or an unconventional moiety.In some embodiments the unconventional moiety is selected from a mirrornucleotide, an abasic ribose moiety and an abasic deoxyribose moiety. Insome embodiments the unconventional moiety is a mirror nucleotide,preferably an L-deoxyribonucleotide (L-DNA) moiety. In some embodimentsat least one of N or N′ includes a 2′-OMe sugar-modified ribonucleotide.

The term “unconventional moiety” as used herein refers to non-nucleotidemoieties including an abasic moiety, an inverted abasic moiety, ahydrocarbon (alkyl) moiety and derivatives thereof, and further includesa deoxyribonucleotide, a modified deoxyribonucleotide, a mirrornucleotide (L-DNA or L-RNA), a non-base pairing nucleotide analog and anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate bond; bridged nucleic acids including LNA and ethylene bridgednucleic acids, linkage modified (e.g. PACE) and base modifiednucleotides, as well as additional moieties explicitly disclosed hereinas unconventional moieties.

As used herein, the term “inhibit”, “down-regulate”, or “reduce” withrespect to gene expression means that the expression of a target gene,or level of RNA molecules or equivalent RNA molecules encoding one ormore proteins or protein subunits (e.g., mRNA), or activity of one ormore proteins or protein subunits, is reduced below that observed in theabsence of an inhibitory factor (such as a nucleic acid molecule, e.g.,an dsNA, for example having structural features as described herein);for example the expression may be reduced to 90%, 80%, 70%, 60%, 50%,40%, 30%, 20%, 10%, 5% or less than that observed in the absence of aninhibitor.

RNA Interference and dsNA Nucleic Acid Molecules

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is often referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing. The processof post-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes (Fire et al., 1999, Trends Genet., 15, 358).Such protection from foreign gene expression may have evolved inresponse to the production of double-stranded RNAs (dsRNAs) derived fromviral infection or from the random integration of transposon elementsinto a host genome via a cellular response that specifically destroyshomologous single-stranded RNA or viral genomic RNA. The presence ofdsRNA in cells triggers the RNAi response through a mechanism that hasyet to be fully characterized. This mechanism appears to be differentfrom other known mechanisms involving double-stranded RNA-specificribonucleases, such as the interferon response that results fromdsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylatesynthetase resulting in non-specific cleavage of mRNA by ribonuclease L(see for example U.S. Pat. Nos. 6,107,094; 5,898,031; Clemens et al.,1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001,Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andinclude about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity.

Nucleic acid molecules (for example having structural features asdisclosed herein) may inhibit or down regulate gene expression or viralreplication by mediating RNA interference “RNAi” or gene silencing in asequence-specific manner; see e.g., Zamore et al., 2000, Cell, 101,25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature,411, 494-498; and Kreutzer et al., International PCT Publication No. WO00/44895; Zernicka-Goetz et al., International PCT Publication No. WO01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetincket al., International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).

A double-stranded nucleic acid molecule can be assembled from twoseparate polynucleotide strands, where one strand is the sense strandand the other is the antisense strand in which the antisense and sensestrands are self-complementary (i.e. each strand includes nucleotidesequence that is complementary to nucleotide sequence in the otherstrand); such as where the antisense strand and sense strand form aduplex or double-stranded structure having any length and structure asdescribed herein for nucleic acid molecules as provided, for examplewherein the double-stranded region (duplex region) is about 15 to about40 (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs); the antisensestrand includes nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule (i.e., TLR2 and TLR4 mRNA) ora portion thereof and the sense strand includes nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof(e.g., about 17 to about 40 nucleotides of the nucleic acid moleculesherein are complementary to the target nucleic acid or a portionthereof).

In certain aspects and embodiments a nucleic acid molecule (e.g., a dsNAmolecule) provided herein may be a “RISC length” molecule or may be aDicer substrate as described in more detail below.

A dsNA nucleic acid molecule may include separate sense and antisensesequences or regions, where the sense and antisense regions arecovalently linked by nucleotide or non-nucleotide linkers molecules asis known in the art, or are alternately non-covalently linked by ionicinteractions, hydrogen bonding, van der Waals interactions, hydrophobicinteractions, and/or stacking interactions. Nucleic acid molecules mayinclude a nucleotide sequence that is complementary to nucleotidesequence of a target gene or of a target mRNA. Nucleic acid moleculesmay interact with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene.

Alternatively, a dsNA nucleic acid molecule is assembled from a singlepolynucleotide, where the self-complementary sense and antisense regionsof the nucleic acid molecules are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s), i.e., the antisense strandand the sense strand are part of one single polynucleotide that havingan antisense region and sense region that fold to form a duplex region(for example to form a “hairpin” structure as is well known in the art).Such dsNA nucleic acid molecules can be a polynucleotide with a duplex,asymmetric duplex, hairpin or asymmetric hairpin secondary structure,having self-complementary sense and antisense regions, wherein theantisense region includes nucleotide sequence that is complementary tonucleotide sequence in a separate target nucleic acid molecule (e.g.TLR2 mRNA or TLR4 mRNA) or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence(i.e., a sequence of TLR2 mRNA or a sequence of TLR4 mRNA). Such dsNAnucleic acid molecules can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion includes nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule (e.g. TLR2 mRNA or TLR4 mRNA)or a portion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence (e.g. TLR2 mRNA orTLR4 mRNA) or a portion thereof, and wherein the circular polynucleotidecan be processed either in vivo or in vitro to generate an activenucleic acid molecule capable of mediating RNAi.

Chemical Modifications of Nucleic Acid Molecules

In certain aspects and embodiments, the methods provided herein utilizesnucleic acid therapeutic agents. The nucleic acid molecules (e.g., dsNAmolecules) as provided herein include one or more modifications (orchemical modifications). Without being bound to theory, the chemicalmodifications confer upon the nucleic acid molecules beneficialproperties including nuclease stability, reduced off-target activity andor reduced immune stimulation. In certain embodiments, suchmodifications include any changes to a nucleic acid molecule orpolynucleotide that would make the molecule different than a standardribonucleotide or RNA molecule (i.e., that includes standard adenine,cytosine, uracil, or guanine moieties); which may be referred to as an“unmodified” ribonucleotide or unmodified ribonucleic acid. TraditionalDNA bases and polynucleotides having a 2′-deoxy sugar represented byadenine, cytosine, thymine, or guanine moieties may be referred to as an“unmodified deoxyribonucleotide” or “unmodified deoxyribonucleic acid”;accordingly, the term “unmodified nucleotide” or “unmodified nucleicacid” as used herein refers to an “unmodified ribonucleotide” or“unmodified ribonucleic acid” unless there is a clear indication to thecontrary. Such modifications can be in the nucleotide sugar, nucleotidebase, nucleotide phosphate group and/or the phosphate backbone of apolynucleotide.

In certain embodiments, modifications as disclosed herein, may be usedto increase RNAi activity of a dsNA molecule and/or to increase the invivo stability of the dsNA molecules, particularly the stability inserum, and/or to increase bioavailability of the dsNA molecules.Non-limiting examples of modifications include without limitationinternucleotide or internucleoside linkages; deoxyribonucleotides ordideoxyribonucleotides at any position and strand of the double-strandednucleic acid molecule; nucleic acid (e.g., ribonucleic acid) with amodification at the 2′-position preferably selected from an amino,fluoro, methoxy, alkoxy and alkyl; 2′-deoxyribonucleotidcs, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, biotingroup, and terminal glyceryl and/or inverted deoxy abasic residueincorporation, sterically hindered molecules, such as fluorescentmolecules and the like. Other nucleotides modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). Further details on variousmodifications are described in more detail below.

Non-limiting examples of chemically modified nucleotides having anorthern configuration include locked nucleic acid (LNA) nucleotides(e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl) nucleotides);2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides. Locked nucleic acids, or LNA'sare described, for example, in Elman et al., 2005; Kurreck et al., 2002;Crinelli et al., 2002; Braasch and Corey, 2001; Bondensgaard et al.,2000; Wahlestedt et al., 2000; and International Patent Publication Nos.WO 00/47599, WO 99/14226, and WO 98/39352 and WO 2004/083430. In oneembodiment of the therapeutic agent provided herein, an LNA isincorporated at the 5′ terminus of the sense strand of the nucleic acidmolecule.

Chemical modifications also include unlocked nucleic acids, or UNAs,which are non-nucleotide, acyclic analogues, in which the C2′-C3′ bondis not present (although UNAs are not truly nucleotides, they areexpressly included in the scope of “modified” nucleotides or modifiednucleic acids as contemplated herein). Exemplary UNAs are disclosed inNucleic Acids Symposium Series No. 52 p. 133-134 (2008). In certainembodiments a nucleic acid molecule (e.g., a siNA molecule) as describedherein include one or more UNAs; or one UNA. In some embodiments, anucleic acid molecule (e.g., a siNA molecule) as described herein has a3′-overhang that includes one or two UNAs in the 3′ overhang. In someembodiments a nucleic acid molecule (e.g., a siNA molecule) as describedherein includes a UNA (for example one UNA) in the antisense strand; forexample in position 6 or position 7 of the antisense strand.

Chemical modifications also include non-pairing nucleotide analogs, forexample as disclosed herein. Chemical modifications further includeunconventional moieties as disclosed herein.

Chemical modifications also include terminal modifications on the 5′and/or 3′ part of the oligonucleotides and are also known as cappingmoieties. Such terminal modifications are selected from a nucleotide, amodified nucleotide, a lipid, a peptide, and a sugar, an abasic ribosemoiety and an abasic deoxyribose moiety.

Chemical modifications also include six membered “six membered ringnucleotide analogs.” Examples of six-membered ring nucleotide analogsare disclosed in Allart, et al (Nucleosides & Nucleotides, 1998,17:1523-1526; and Perez-Perez, et al., 1996, Bioorg. and Medicinal ChemLetters 6:1457-1460) Oligonucleotides including 6-membered ringnucleotide analogs including hexitol and altritol nucleotide monomersare disclosed in International patent application publication No. WO2006/047842.

Chemical modifications also include “mirror” nucleotides which have areversed chirality as compared to normal naturally occurring nucleotide;that is a mirror nucleotide may be an “L-nucleotide” analogue ofnaturally occurring D-nucleotide (see U.S. Pat. No. 6,602,858). Mirrornucleotides may further include at least one sugar or base modificationand/or a backbone modification, for example, as described herein, suchas a phosphorothioate or phosphonate moiety. U.S. Pat. No. 6,602,858discloses nucleic acid catalysts including at least one L-nucleotidesubstitution. Mirror nucleotides include for example L-DNA(L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror image dT)) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouracil-3′-phosphate (mirror dU).

In some embodiments, modified ribonucleotides include modifieddeoxyribonucleotides, for example 5′OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate.

Modifications may be present in one or more strands of a nucleic acidmolecule disclosed herein, e.g., in the sense strand, the antisensestrand, or both strands. In certain embodiments, the antisense strandmay include modifications and the sense strand my only includeunmodified ribonucleotides.

Nucleobases

Nucleobases of the nucleic acid disclosed herein may include unmodifiedribonucleotides (purines and pyrimidines) such as adenine, guanine,cytosine, uridine. The nucleobases in one or both strands can bemodified with natural and synthetic nucleobases such as, thymine,xanthine, hypoxanthine, ionosine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, any “universal base”nucleotides; 2-propyl and other alkyl derivatives of adenine andguanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, deazapurines,heterocyclic substituted analogs of purines and pyrimidines, e.g.,aminoethyoxy phenoxazine, derivatives of purines and pyrimidines (e.g.,1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) andtautomers thereof, 8-oxo-N⁶-methyladenine, 7-diazaxanthine,5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and 4,4-ethanocytosine). Other examples of suitable basesinclude non-purinyl and non-pyrimidinyl bases such as 2-aminopyridineand triazines.

Sugar Moieties

Sugar moieties in nucleic acid disclosed herein may include2′-hydroxyl-pentofuranosyl sugar moiety without any modification.Alternatively, sugar moieties can be modified such as,2′-deoxy-pentofuranosyl sugar moiety, D-ribose, hexose, modification atthe 2′ position of the pentofuranosyl sugar moiety such as 2′-O-alkyl(including 2′-O-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino,2′-O-allyl, 2′-S-alkyl, 2′-halogen (including 2′-fluoro, chloro, andbromo), 2′-methoxyethoxy, 2′-O-methoxyethyl, 2′-O-2-methoxyethyl,2′-allyloxy (—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl,propenyl, CF, cyano, imidazole, carboxylate, thioate, C₁ to C₁₀ loweralkyl, substituted lower alkyl, alkaryl or aralkyl, OCF₃, OCN, O—, S—,or N-alkyl; O—, S, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃;heterozycloalkyl; heterozycloalkaryl; aminoalkylamino; polyalkylamino orsubstituted silyl, as, among others, for example as described inEuropean patents EP 0 586 520 B1 or EP 0 618 925 B1.

Alkyl group includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has 6or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably 4 or fewer. Likewise,preferred cycloalkyls may have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing 1 to 6 carbonatoms. The alkyl group can be substituted alkyl group such as alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Alkoxy group includes substituted and unsubstituted alkyl, alkenyl, andalkynyl groups covalently linked to an oxygen atom. Examples of alkoxygroups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, andpentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

In some embodiments, the pentafuronosyl ring may be replaced withacyclic derivatives lacking the C2′-C3′-bond of the pentafuronosyl ring.For example, acyclonucleotides may substitute a 2-hydroxyethoxymethylgroup for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs.

Halogens include fluorine, bromine, chlorine, iodine.

Backbone

The nucleoside subunits of the nucleic acid disclosed herein may belinked to each other by phosphodiester bond. The phosphodiester bond maybe optionally substituted with other linkages. For example,phosphorothioate, thiophosphate-D-ribose entities, triester, thioate,2′-5′ bridged backbone (may also be referred to as 5′-2′), PACE, 3′-(or-5′)deoxy-3′-(or -5′)thio-phosphorothioate, phosphorodithioate,phosphoroselenates, 3′-(or -5′)deoxy phosphinates, borano phosphates,3′-(or -5′)deoxy-3′-(or -5′)amino phosphoramidates, hydrogenphosphonates, phosphonates, borano phosphate esters, phosphoramidates,alkyl or aryl phosphonates and phosphotriester modifications such asalkylphosphotriesters, phosphotriester phosphorus linkages,5′-ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate,and nonphosphorus containing linkages for example, carbonate, carbamate,silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetyl,oxime, methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino linkages.

Nucleic acid molecules disclosed herein may include a peptide nucleicacid (PNA) backbone. The PNA backbone includes repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The variousbases such as purine, pyrimidine, natural and synthetic bases are linkedto the backbone by methylene carbonyl bonds.

Terminal Phosphates

Modifications can be made at terminal phosphate groups. Non-limitingexamples of different stabilization chemistries can be used, e.g., tostabilize the 3′-end of nucleic acid sequences, including (1)[3-3′]-inverted deoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide.Exemplary chemically modified terminal phosphate groups include thoseshown below:

Conjugates

Modified nucleotides and nucleic acid molecules (e.g., dsNA molecules)as provided herein may include conjugates, for example, a conjugatecovalently attached to the chemically-modified nucleic acid molecule.Non-limiting examples of conjugates include conjugates and ligandsdescribed in Vargeese et al., U.S. Ser. No. 10/427,160. The conjugatemay be covalently attached to a nucleic acid molecule (such as an siNAmolecule) via a biodegradable linker. The conjugate molecule may beattached at the 3′-end of either the sense strand, the antisense strand,or both strands of the chemically-modified nucleic acid molecule. Theconjugate molecule may be attached at the 5′-end of either the sensestrand, the antisense strand, or both strands of the chemically-modifiednucleic acid molecule. The conjugate molecule may be attached both the3′-end and 5′-end of either the sense strand, the antisense strand, orboth strands of the chemically-modified nucleic acid molecule, or anycombination thereof. In one embodiment, a conjugate molecule may includea molecule that facilitates delivery of a chemically-modified nucleicacid molecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiednucleic acid molecule is a polyethylene glycol, human serum albumin, ora ligand for a cellular receptor that can mediate cellular uptake.Examples of specific conjugate molecules contemplated herein that can beattached to chemically-modified nucleic acid molecules are described inVargeese et al., U.S. Ser. No. 10/201,394.

Linkers

A nucleic acid molecule provided herein (e.g., an dsNA) molecule mayinclude a nucleotide, non-nucleotide, or mixed nucleotide/non-nucleotidelinker that joins the sense region of the nucleic acid to the antisenseregion of the nucleic acid. A nucleotide linker can be a linker of ≧2nucleotides in length, for example about 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides in length. The nucleotide linker can be a nucleic acidaptamer. The term “aptamer” or “nucleic acid aptamer” as used hereinrefers to a nucleic acid molecule that binds specifically to a targetmolecule wherein the nucleic acid molecule has sequence that includes asequence recognized by the target molecule in its natural setting.Alternately, an aptamer can be a nucleic acid molecule that binds to atarget molecule (such as TLR2 mRNA and TLR4 mRNA) where the targetmolecule does not naturally bind to a nucleic acid. For example, theaptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. See e.g., Gold et al.; 1995, AnnuRev. Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5;Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol.,74, 27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999,Clinical Chemistry, 45, 1628.

A non-nucleotide linker may include an abasic nucleotide, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, orother polymeric compounds (e.g. polyethylene glycols such as thosehaving between 2 and 100 ethylene glycol units). Specific examplesinclude those described by Seela and Kaiser, Nucleic Acids Res. 1990,18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J.Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem.Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 andBiochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990,18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschkeet al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991,30:9914; Arnold et al., International Publication No. WO 89/02439; Usmanet al., International Publication No. WO 95/06731; Dudycz et al.,International Publication No. WO 95/11910 and Ferentz and Verdine, J.Am. Chem. Soc. 1991, 113:4000.

5′ Ends, 3′ Ends and Overhangs

Nucleic acid molecules disclosed herein (e.g., dsNA molecules) may beblunt-ended on both sides, have overhangs on both ends or a combinationof blunt and overhang ends. Overhangs may occur on either the 5′- or3′-end of the sense or antisense strand.

5′- and/or 3′-ends of double-stranded nucleic acid molecules (e.g.,dsNA) may be blunt ended or have an overhang. The 5′-end may be bluntended and the 3′-end has an overhang in either the sense strand or theantisense strand. In other embodiments, the 3′-end may be blunt endedand the 5′-end has an overhang in either the sense strand or theantisense strand. In yet other embodiments, both the 5′- and 3′-end areblunt ended or both the 5′- and 3′-ends have overhangs.

The 5′- and/or 3′-end of one or both strands of the nucleic acid mayinclude a free hydroxyl group. The 5′- and/or 3′-end of any nucleic acidmolecule strand may be modified to include a chemical modification. Suchmodification may stabilize nucleic acid molecules, e.g., the 3′-end mayhave increased stability due to the presence of the nucleic acidmolecule modification. Examples of end modifications (e.g., terminalcaps) include, but are not limited to, abasic, deoxy abasic, inverted(deoxy) abasic, glyceryl, dinucleotide, acyclic nucleotide, amino,fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate,C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl,OCF₃, OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂, N₃; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino or substituted silyl, as, among others, described inEuropean patents EP 586,520 and EP 618,925 and other modificationsdisclosed herein.

Nucleic acid molecules include those with blunt ends, i.e., ends that donot include any overhanging nucleotides. A nucleic acid molecule caninclude one or more blunt ends. The blunt ended nucleic acid moleculehas a number of base pairs equal to the number of nucleotides present ineach strand of the nucleic acid molecule. The nucleic acid molecule caninclude one blunt end, for example where the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. Nucleic acid molecule may include one blunt end, forexample where the 3′-end of the antisense strand and the 5′-end of thesense strand do not have any overhanging nucleotides. A nucleic acidmolecule may include two blunt ends, for example where the 3′-end of theantisense strand and the 5′-end of the sense strand as well as the5′-end of the antisense strand and 3′-end of the sense strand do nothave any overhanging nucleotides. Other nucleotides present in a bluntended nucleic acid molecule can include, for example, mismatches,bulges, loops, or wobble base pairs to modulate the activity of thenucleic acid molecule, e.g. to mediate RNA interference.

In certain embodiments of the nucleic acid molecules (e.g., dsNAmolecules) provided herein, at least one end of the molecule has anoverhang of at least one nucleotide (for example 1 to 8 overhangnucleotides). For example, one or both strands of a double-strandednucleic acid molecule disclosed herein may have an overhang at the5′-end or at the 3′-end or both. An overhang may be present at either orboth the sense strand and antisense strand of the nucleic acid molecule.The length of the overhang may be as little as one nucleotide and aslong as 1 to 8 or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7 or 8nucleotides; in some preferred embodiments an overhang is 2, 3, 4, 5, 6,7 or 8 nucleotides; for example an overhang may be 2 nucleotides. Thenucleotide(s) forming the overhang may be includedeoxyribonucleotide(s), ribonucleotide(s), natural and non-naturalnucleobases or any nucleotide modified in the sugar, base or phosphategroup, such as disclosed herein. A double-stranded nucleic acid moleculemay have both 5′- and 3′-overhangs. The overhangs at the 5′- and 3′-endmay be of different lengths. A overhang may include at least one nucleicacid modification which may be deoxyribonucleotide. One or moredeoxyribonucleotides may be at the 5′-terminus. The 3′-end of therespective counter-strand of the nucleic acid molecule may not have anoverhang, more preferably not a deoxyribonucleotide overhang. The one ormore deoxyribonucleotide may be at the 3′-terminus. The 5′-end of therespective counter-strand of the dsRNA may not have an overhang, morepreferably not a deoxyribonucleotide overhang. The overhang in eitherthe 5′- or the 3′-end of a strand may be 1 to 8 (e.g., about 1, 2, 3, 4,5, 6, 7 or 8) unpaired nucleotides, preferably, the overhang is 2-3unpaired nucleotides; more preferably 2 unpaired nucleotides. Nucleicacid molecules may include duplex nucleic acid molecules withoverhanging ends of about 1 to about 20 (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19 or 20); preferably 1-8(e.g., about 1, 2, 3, 4, 5, 6, 7 or 8) nucleotides, for example, about21-nucleotide duplexes with about 19 base pairs and 3′-terminalmononucleotide, dinucleotide, or trinucleotide overhangs. Nucleic acidmolecules provided herein may include duplex nucleic acid molecules withblunt ends, where both ends are blunt, or alternatively, where one ofthe ends is blunt. Nucleic acid molecules disclosed herein can includeone or more blunt ends, i.e. where a blunt end does not have anyoverhanging nucleotides. In one embodiment, the blunt ended nucleic acidmolecule has a number of base pairs equal to the number of nucleotidespresent in each strand of the nucleic acid molecule. The nucleic acidmolecule may include one blunt end, for example where the 5′-end of theantisense strand and the 3′-end of the sense strand do not have anyoverhanging nucleotides. The nucleic acid molecule may include one bluntend, for example where the 3′-end of the antisense strand and the 5′-endof the sense strand do not have any overhanging nucleotides. A nucleicacid molecule may include two blunt ends, for example where the 3′-endof the antisense strand and the 5′-end of the sense strand as well asthe 5′-end of the antisense strand and 3′-end of the sense strand do nothave any overhanging nucleotides. In certain preferred embodiments thenucleic acid compounds are blunt ended. Other nucleotides present in ablunt ended dsNA molecule can include, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the nucleic acidmolecule to mediate RNA interference.

In many embodiments one or more, or all, of the overhang nucleotides ofa nucleic acid molecule (e.g., a dsNA molecule) as described hereinincludes are modified such as described herein; for example one or more,or all, of the nucleotides may be 2′-deoxynucleotides.

Amount, Location and Patterns of Modifications of Nucleic Acid Compounds

Nucleic acid molecules (e.g., dsNA molecules) disclosed herein mayinclude modified nucleotides as a percentage of the total number ofnucleotides present in the nucleic acid molecule. As such, a nucleicacid molecule may include about 5% to about 100% modified nucleotides(e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). Theactual percentage of modified nucleotides present in a given nucleicacid molecule will depend on the total number of nucleotides present inthe nucleic acid. If the nucleic acid molecule is single stranded, thepercent modification can be based upon the total number of nucleotidespresent in the single stranded nucleic acid molecule. Likewise, if thenucleic acid molecule is double-stranded, the percent modification canbe based upon the total number of nucleotides present in the sensestrand, antisense strand, or both the sense and antisense strands.

Nucleic acid molecules disclosed herein may include unmodified RNA as apercentage of the total nucleotides in the nucleic acid molecule. Assuch, a nucleic acid molecule may include about 5% to about 100%unmodified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% oftotal nucleotides present in a nucleic acid molecule).

A nucleic acid molecule (e.g., an dsNA molecule) may include a sensestrand that includes about 1 to about 5, specifically about 1, 2, 3, 4,or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand includes about 1 to about5 or more, specifically about 1, 2, 3, 4, 5, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. A nucleic acid molecule may include about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the senseand/or antisense nucleic acid strand are chemically-modified with2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with orwithout about 1 to about 5 or more, for example about 1, 2, 3, 4, 5, ormore phosphorothioate internucleotide linkages and/or a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends,being present in the same or different strand.

A nucleic acid molecule may include about 1 to about 5 or more(specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the nucleic acid molecule.

A nucleic acid molecule may include 2′-5′ internucleotide linkages, forexample at the 3′-end, the 5′-end, or both of the 3′-end and 5′-end ofone or both nucleic acid sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both nucleic acid sequence strands, for example, about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotidelinkage of a pyrimidine nucleotide in one or both strands of the siNAmolecule can include a 2′-5′ internucleotide linkage, or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage ofa purine nucleotide in one or both strands of the siNA molecule caninclude a 2′-5′ internucleotide linkage.

A chemically-modified short interfering nucleic acid (dsNA) molecule mayinclude an antisense region, wherein any (e.g., one or more or all)pyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides).

A chemically-modified short interfering nucleic acid (dsNA) molecule mayinclude an antisense region, wherein any (e.g., one or more or all)pyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides).

A chemically-modified short interfering nucleic acid (dsNA) moleculecapable of mediating RNA interference (RNAi) against TLR2 and/or TLR4inside a cell or reconstituted in vitro system may include a senseregion, wherein one or more pyrimidine nucleotides present in the senseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides), and anantisense region, wherein one or more pyrimidine nucleotides present inthe antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides(e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides or alternately a plurality of pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and one ormore purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modification,that is optionally present at the 3′-end, the 5′-end, or both of the3′-end and the 5′-end of the sense and/or antisense sequence. The senseand/or antisense region can optionally further include a 3′-terminalnucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or4) 2′-deoxyribonucleotides. The overhang nucleotides can further includeone or more (e.g., about 1, 2, 3, 4 or more) phosphorothioate,phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages.The purine nucleotides in the sense region may alternatively be2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides) and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). One or more purine nucleotides in thesense region may alternatively be purine ribonucleotides (e.g., whereinall purine nucleotides are purine ribonucleotides or alternately aplurality of purine nucleotides are purine ribonucleotides) and anypurine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). One or more purine nucleotides in thesense region and/or present in the antisense region may alternatively beselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein allpurine nucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides oralternately a plurality of purine nucleotides are selected from thegroup consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides).

In some embodiments, a nucleic acid molecule (e.g., a dsNA molecule) asdescribed herein includes a modified nucleotide (for example onemodified nucleotide) in the antisense strand; for example in position 6or position 7 of the antisense strand.

Modification Patterns and Alternating Modifications of Nucleic AcidCompounds

Nucleic acid molecules (e.g., dsNA molecules) provided herein may havepatterns of modified and unmodified nucleic acids. A pattern ofmodification of the nucleotides in a contiguous stretch of nucleotidesmay be a modification contained within a single nucleotide or group ofnucleotides that are covalently linked to each other via standardphosphodiester bonds or, at least partially, through phosphorothioatebonds. Accordingly, a “pattern” as contemplated herein, does notnecessarily need to involve repeating units, although it may. Examplesof modification patterns that may be used in conjunction with thenucleic acid molecules (e.g., dsNA molecules) provided herein includethose disclosed in Giese, U.S. Pat. No. 7,452,987. For example, nucleicacid molecules (e.g., dsNA molecules) provided herein include thosehaving modification patterns such as, similar to, or the same as, thepatterns shown diagrammatically in FIG. 2 of the Giese U.S. Pat. No.7,452,987.

A modified nucleotide or group of modified nucleotides may be at the5′-end or the 3′-end of the sense strand or the antisense strand, aflanking nucleotide or group of nucleotides is arrayed on both sides ofthe modified nucleotide or group, where the flanking nucleotide or groupeither is unmodified or does not have the same modification of thepreceding nucleotide or group of nucleotides. The flanking nucleotide orgroup of nucleotides may, however, have a different modification. Thissequence of modified nucleotide or group of modified nucleotides,respectively, and unmodified or differently modified nucleotide or groupof unmodified or differently modified nucleotides may be repeated one ormore times.

In some patterns, the 5′-terminal nucleotide of a strand is a modifiednucleotide while in other patterns the 5′-terminal nucleotide of astrand is an unmodified nucleotide. In some patterns, the 5′-end of astrand starts with a group of modified nucleotides while in otherpatterns, the 5′-terminal end is an unmodified group of nucleotides.This pattern may be either on the first stretch or the second stretch ofthe nucleic acid molecule or on both.

Modified nucleotides of one strand of the nucleic acid molecule may becomplementary in position to the modified or unmodified nucleotides orgroups of nucleotides of the other strand.

There may be a phase shift between modifications or patterns ofmodifications on one strand relative to the pattern of modification ofthe other strand such that the modification groups do not overlap. Inone instance, the shift is such that the modified group of nucleotidesof the sense strand corresponds to the unmodified group of nucleotidesof the antisense strand and vice versa.

There may be a partial shift of the pattern of modification such thatthe modified groups overlap. The groups of modified nucleotides in anygiven strand may optionally be the same length, but may be of differentlengths. Similarly, groups of unmodified nucleotides in any given strandmay optionally be the same length, or of different lengths.

In some patterns, the second (penultimate) nucleotide at the terminus ofthe strand, is an unmodified nucleotide or the beginning of group ofunmodified nucleotides. Preferably, this unmodified nucleotide orunmodified group of nucleotides is located at the 5′-end of the eitheror both the sense strand and the antisense strand and even morepreferably at the terminus of the sense strand. An unmodified nucleotideor unmodified group of nucleotide may be located at the 5′-end of thesense strand. In one embodiment the pattern consists of alternatingsingle modified and unmodified nucleotides.

In some double-stranded nucleic acid molecules a 2′-O-methyl modifiednucleotide and a non-modified nucleotide or a nucleotide which is not2′-O-methyl modified, are incorporated on both strands in an alternatingfashion, resulting in a pattern of alternating 2′-O-methyl modifiednucleotides and nucleotides that are either unmodified or at least donot include a 2′-O-methyl modification. In certain embodiments, the samesequence of 2′-O-methyl modification and non-modification exists on thesecond strand; in other embodiments the alternating 2′-O-methyl modifiednucleotides are only present in the sense strand and are not present inthe antisense strand; and in yet other embodiments the alternating2′-O-methyl modified nucleotides are only present in the antisensestrand and are not present in the sense strand. In certain embodiments,there is a phase shift between the two strands such that the 2′-O-methylmodified nucleotide on the first strand base pairs with a non-modifiednucleotide(s) on the second strand and vice versa. This particulararrangement, i.e. base pairing of 2′-O-methyl modified and non-modifiednucleotide(s) on both strands is particularly preferred in certainembodiments. In certain embodiments, the pattern of alternating2′-O-methyl modified nucleotides exists throughout the entire nucleicacid molecule; or the entire duplex region. In other embodiments thepattern of alternating 2′-O-methyl modified nucleotides exists only in aportion of the nucleic acid; or portion of the duplex region.

In “phase shift” patterns, it may be preferred if the antisense strandstarts with a 2′-O-methyl modified nucleotide at the 5′ end wherebyconsequently the second nucleotide is non-modified, the third, fifth,seventh and so on nucleotides are thus again 2′-O-methyl modifiedwhereas the second, fourth, sixth, eighth and the like nucleotides arenon-modified nucleotides.

Exemplary Modification Locations and Patterns of Nucleic Acid Compounds

While exemplary patterns are provided in more detail below, allpermutations of patterns with all possible characteristics of thenucleic acid molecules disclosed herein and those known in the art arecontemplated (e.g., characteristics include, but are not limited to,length of sense strand, length of antisense strand, length of duplexregion, length of hangover, whether one or both ends of adouble-stranded nucleic acid molecule is blunt or has an overhang,location of modified nucleic acid, number of modified nucleic acids,types of modifications, whether a double overhang nucleic acid moleculehas the same or different number of nucleotides on the overhang of eachside, whether a one or more than one type of modification is used in anucleic acid molecule, and number of contiguous modified/unmodifiednucleotides). With respect to all detailed examples provided below,while the duplex region is shown to be 19 nucleotides, the nucleic acidmolecules provided herein can have a duplex region ranging from 1 to 40nucleotides in length as each strand of a duplex region canindependently be 17-40 nucleotides in length Exemplary patterns areprovided herein.

Nucleic acid molecules may have a blunt end on both ends that include asingle or contiguous set of modified nucleic acids. The modified nucleicacid may be located at any position along either the sense or antisensestrand. Nucleic acid molecules may include a group of about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40contiguous modified nucleotides. Modified nucleic acids may make up 1%,2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 100% of a nucleic acid strand.Modified nucleic acids of the examples immediately below may be in thesense strand only, the antisense strand only, or in both the sensestrand and the antisense strand.

Nicks and Gaps in Nucleic Acid Strands

Nucleic acid molecules (e.g., siNA molecules) provided herein may have astrand, preferably the sense strand, that is nicked or gapped. As such,nucleic acid molecules may have three or more strand, for example, suchas a meroduplex RNA (mdRNA) disclosed in International PatentApplication No. PCT/US07/081836. Nucleic acid molecules with a nicked orgapped strand may be RISC length (e.g., about 15 to 25 nucleotides) orDicer substrate length (e.g., about 25 to 30 nucleotides).

Dicer Substrates

In certain embodiments, the nucleic acid molecules (e.g., siNAmolecules) provided herein may be a precursor “Dicer substrate”molecule, e.g., double-stranded nucleic acid, processed in vivo toproduce an active nucleic acid molecules, for example as described inRossi, US Patent App. No. 20050244858. In certain conditions andsituations, it has been found that these relatively longer dsRNA siNAspecies, e.g., of from about 25 to about 30 nucleotides, can giveunexpectedly effective results in terms of potency and duration ofaction. Without wishing to be bound by any particular theory, it isthought that the longer dsRNA species serve as a substrate for theenzyme Dicer in the cytoplasm of a cell. In addition to cleavingdouble-stranded nucleic acid into shorter segments, Dicer may facilitatethe incorporation of a single-stranded cleavage product derived from thecleaved dsRNA into the RNA-induced silencing complex (RISC complex) thatis responsible for the destruction of the cytoplasmic RNA derived fromthe target gene.

Dicer substrates may have certain properties which enhance itsprocessing by Dicer. Dicer substrates are of a length sufficient suchthat it is processed by Dicer to produce an active nucleic acid moleculeand may further include one or more of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′ overhang on the first strand(antisense strand) and (ii) the dsRNA has a modified 3′ end on thesecond strand (sense strand) to direct orientation of Dicer binding andprocessing of the dsRNA to an active siRNA. In certain embodiments, thelongest strand in the Dicer substrate may be 24-30 nucleotides.

Dicer substrates may be symmetric or asymmetric. The Dicer substrate mayhave a sense strand that includes 22-28 nucleotides and an antisensestrand that may include 24-30 nucleotides; thus, in some embodiments theresulting Dicer substrate may have an overhang on the 3′ end of theantisense strand. Dicer substrate may have a sense strand 25 nucleotidesin length, and an antisense strand having 27 nucleotides in length witha 3′-overhang. The overhang may be 1-3 nucleotides, for example 2nucleotides. The sense strand may also have a 5′ phosphate.

Like other siNA molecules provided herein, the antisense strand of aDicer substrate may have any sequence that anneals to the antisensestrand under biological conditions, such as within the cytoplasm of aeukaryotic cell.

Dicer substrates may have any modifications to the nucleotide base,sugar or phosphate backbone as known in the art and/or as describedherein for other nucleic acid molecules (such as siNA molecules). Incertain embodiments, Dicer substrates may have a sense strand that ismodified for Dicer processing by suitable modifiers located at the 3′end of the sense strand, i.e., the dsRNA is designed to directorientation of Dicer binding and processing. Suitable modifiers includenucleotides such as deoxyribonucleotides, dideoxyribonucleotides,acyclonucleotides and the like and sterically hindered molecules, suchas fluorescent molecules and the like. Acyclonucleotides substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normallypresent in deoxynucleoside monophosphates (dNMPs). Other nucleotidemodifiers that could be used in Dicer substrate siNA molecules include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, they may replace ribonucleotides (e.g., 1-3 nucleotidemodifiers, or 2 nucleotide modifiers are substituted for theribonucleotides on the 3′ end of the sense strand) such that the lengthof the Dicer substrate does not change. When sterically hinderedmolecules are utilized, they may be attached to the ribonucleotide atthe 3′ end of the antisense strand. Thus, in certain embodiments thelength of the strand does not change with the incorporation of themodifiers. In certain embodiments, two DNA bases in the dsRNA aresubstituted to direct the orientation of Dicer processing of theantisense strand. In a further embodiment, two terminal DNA bases aresubstituted for two ribonucleotides on the 3′-end of the sense strandforming a blunt end of the duplex on the 3′ end of the sense strand andthe 5′ end of the antisense strand, and a two-nucleotide RNA overhang islocated on the 3′-end of the antisense strand. This is an asymmetriccomposition with DNA on the blunt end and RNA bases on the overhangingend.

In certain embodiments modifications are included in the Dicer substratesuch that the modification does not prevent the nucleic acid moleculefrom serving as a substrate for Dicer. In one embodiment, one or moremodifications are made that enhance Dicer processing of the Dicersubstrate. One or more modifications may be made that result in moreeffective RNAi generation. One or more modifications may be made thatsupport a greater RNAi effect. One or more modifications are made thatresult in greater potency per each Dicer substrate to be delivered tothe cell. Modifications may be incorporated in the 3′-terminal region,the 5′-terminal region, in both the 3′-terminal and 5′-terminal regionor at various positions within the sequence. Any number and combinationof modifications can be incorporated into the Dicer substrate so long asthe modification does not prevent the nucleic acid molecule from servingas a substrate for Dicer. Where multiple modifications are present, theymay be the same or different. Modifications to bases, sugar moieties,the phosphate backbone, and their combinations are contemplated. Either5′-terminus can be phosphorylated.

The sense and antisense strands of the Dicer substrate are not requiredto be completely complementary. They only need to be substantiallycomplementary to anneal under biological conditions and to provide asubstrate for Dicer that produces an siRNA sufficiently complementary tothe target sequence.

A region of one of the strands, particularly the antisense strand, ofthe Dicer substrate may have a sequence length of at least 19nucleotides, wherein these nucleotides are in the 21-nucleotide regionadjacent to the 3′ end of the antisense strand and are sufficientlycomplementary to a nucleotide sequence of the RNA produced from thetarget gene. A Dicer substrate may also have one or more of thefollowing additional properties: (a) the antisense strand has a rightshift from a corresponding 21-mer (i.e., the antisense strand includesnucleotides on the right side of the molecule when compared to thecorresponding 21-mer), (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairingsand (c) base modifications such as locked nucleic acid(s) may beincluded in the 5′ end of the sense strand.

An antisense strand of a Dicer substrate nucleic acid molecule may bemodified to include 1-9 ribonucleotides on the 5′-end to give a lengthof 22-28 nucleotides. When the antisense strand has a length of 21nucleotides, then 1-7 ribonucleotides, or 2-5 ribonucleotides and or 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense strands is not required. That is,the resultant antisense strand is sufficiently complementary with thetarget sequence. A sense strand may then have 24-30 nucleotides. Thesense strand may be substantially complementary with the antisensestrand to anneal to the antisense strand under biological conditions. Inone embodiment, the antisense strand may be synthesized to contain amodified 3′-end to direct Dicer processing. The sense strand may have a3′ overhang. The antisense strand may be synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strandmay have a 3′ overhang.

Methods and Compositions for Inhibiting TLR2 and TLR4

In various aspects provided are compositions and methods for inhibitionof TLR2 expression for treatment of lung disease, disorder or injury ina mammal. In various embodiments the method comprises administering tothe mammal at least one therapeutic agent selected from a TLR2 inhibitoror a pharmaceutically acceptable salt or prodrug thereof; in an amounteffect to treat the mammal. In various embodiments the therapeutic agentis selected from the group consisting of a small molecule chemicalcompound; a protein; an antibody or fragment thereof; and a nucleic acidmolecule.

In various aspects provided are compositions and methods for inhibitionof TLR2 and TLR4 expression for the treatment of lung disease, disorderor injury in a mammal. In various embodiments the method comprisesadministering to the mammal at least two therapeutic agents selectedfrom: (i) a TLR2 inhibitor or a pharmaceutically acceptable salt orprodrug thereof and (ii) a TLR4 inhibitor or a pharmaceuticallyacceptable salt or prodrug thereof; in an amount effective to treat themammal. In various embodiments each therapeutic agent is independentlyselected from the group consisting of a small molecule chemicalcompound; a protein; an antibody or fragment thereof; and a nucleic acidmolecule.

In some embodiments, the therapeutic agent is a combined inhibitor bywhich it is meant a single agent which is capable of inhibiting theexpression and/or activity of at least two genes and/or gene products ofboth: TLR2 and TLR4. Non-limiting examples of such single agents aretandem and multi-armed RNAi molecules disclosed in PCT PatentPublication No. WO 2007/091269.

In some embodiments a small nucleic acid molecule is selected from ashort interfering nucleic acid (siNA), double-stranded nucleic acid(dsNA), interfering RNA (RNAi), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules capable of mediating or that mediate RNA interferenceagainst TLR2 and TLR4 gene expression. The composition and methodsdisclosed herein are also useful in treating or preventing inflammationand/or graft rejection associated with organ transplantation, inparticular lung transplantation, including treatment, prevention orattenuation of progression of primary graft failure,ischemia-reperfusion injury, reperfusion injury, reperfusion edema,allograft dysfunction, pulmonary reimplantation response, bronchiolitisobliterans after lung transplantation and/or primary graft dysfunction(PGD) after organ transplantation, in particular lung transplantation.

Nucleic acid molecule(s) and/or methods provided herein may be used todown regulate the expression of gene(s) that encode RNA referred to, byexample, Genbank Accession numbers NM_003264.3 (TLR2), NR_024169.1(TLR4), NM_138554.3 (TLR4) and NR_024168.1 (TLR4).

Compositions, methods and kits provided herein may include one or morenucleic acid molecules (e.g., dsNA) and methods that independently or incombination modulate (e.g., down-regulate) the expression of TLR2 andTLR4 protein and/or genes encoding TLR2 and TLR4 proteins associatedwith the maintenance and/or development of diseases, conditions ordisorders such as acute respiratory distress syndrome (ARDS), acute lunginjury, pulmonary fibrosis (idiopathic), bleomycin induced pulmonaryfibrosis, mechanical ventilation induced lung injury, chronicobstructive pulmonary disease (COPD), chronic bronchitis, emphysema,primary graft failure, ischemia-reperfusion injury, reperfusion injury,reperfusion edema, allograft dysfunction, pulmonary reimplantationresponse, bronchiolitis obliterans after lung transplantation and/orprimary graft dysfunction (PGD) after organ transplantation, inparticular in lung transplant (e.g., genes encoding sequences comprisingthose sequences referred to by GenBank Accession Nos. NM_003264.3,NR_024169.1, NM_138554.3 and NR_024168.1, or a TLR2 and TLR4 gene familymember where the genes or gene family sequences share sequencehomology). The description of the various aspects and embodiments isprovided with reference to exemplary genes TLR2 and TLR4. However, thevarious aspects and embodiments are also directed to other related TLR2and TLR4 genes, such as homolog genes and transcript variants, andpolymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associatedwith certain TLR2 and TLR4 genes. As such, the various aspects andembodiments are also directed to other genes that are involved in TLR2and TLR4 mediated pathways of signal transduction or gene expressionthat are involved, for example, in the maintenance or development ofdiseases, traits, or conditions described herein. These additional genescan be analyzed for target sites using the methods described for theTLR2 and TLR4 genes herein. Thus, the modulation of other genes and theeffects of such modulation of the other genes can be performed,determined, and measured as described herein.

In one embodiment, compositions and methods provided herein include adouble-stranded short interfering nucleic acid (dsNA) molecule thatdown-regulates expression of TLR2 gene (e.g., human TLR2 exemplified bySEQ ID NO:1), where the nucleic acid molecule includes about 17 to about40 base pairs.

In one embodiment, compositions and methods provided herein include adouble-stranded short interfering nucleic acid (dsNA) molecules thatdown-regulates expression of TLR2 gene and TLR4 gene (e.g., human TLR2exemplified by SEQ ID NO:1 and human TLR4 exemplified by SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:4), where the nucleic acid molecules includesabout 17 to about 40 base pairs.

In one embodiment, a nucleic acid disclosed herein may be used toinhibit the expression of the TLR2 and/or TLR4 gene or a TLR2 and/orTLR4 gene family where the genes or gene family sequences share sequencehomology. Such homologous sequences can be identified as is known in theart, for example using sequence alignments. Nucleic acid molecules canbe designed to target such homologous sequences, for example usingperfectly complementary sequences or by incorporating non-canonical basepairs, for example mismatches and/or wobble base pairs, that can provideadditional target sequences. In instances where mismatches areidentified, non-canonical base pairs (for example, mismatches and/orwobble bases) can be used to generate nucleic acid molecules that targetmore than one gene sequence. In a non-limiting example, non-canonicalbase pairs such as UU and CC base pairs are used to generate nucleicacid molecules that are capable of targeting sequences for differingTLR2 and/or TLR4 targets that share sequence homology. As such, oneadvantage of using dsRNAs disclosed herein is that a single nucleic acidcan be designed to include nucleic acid sequence that is complementaryto the nucleotide sequence that is conserved between the homologousgenes. In this approach, a single nucleic acid can be used to inhibitexpression of more than one gene instead of using more than one nucleicacid molecule to target the different genes.

Nucleic acid molecules may be used to target conserved sequencescorresponding to a gene family or gene families such as TLR2 and/or TLR4family genes. As such, nucleic acid molecules targeting multiple TLR2and/or TLR4 targets can provide increased therapeutic effect. Inaddition, nucleic acid can be used to characterize pathways of genefunction in a variety of applications. For example, nucleic acidmolecules can be used to inhibit the activity of target gene(s) in apathway to determine the function of uncharacterized gene(s) in genefunction analysis, mRNA function analysis, or translational analysis.The nucleic acid molecules can be used to determine potential targetgene pathways involved in various diseases and conditions towardpharmaceutical development. The nucleic acid molecules can be used tounderstand pathways of gene expression involved in, for example acuterespiratory distress syndrome (ARDS), acute lung injury, pulmonaryfibrosis (idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilation induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, emphysema, bronchiolitis obliterans afterlung transplantation, and/or inflammation and/or graft rejection,associated with organ transplantation-induced acute graft dysfunction,in particular lung transplantation-induced acute graft dysfunction.

In one embodiment, the compositions and methods provided herein includea nucleic acid molecule having RNAi activity against TLR2. In anotherembodiment, the compositions and methods provided herein include anucleic acid molecule having RNAi activity against TLR2 RNA and anucleic acid molecule having RNAi activity against TLR4 RNA, where thenucleic acid molecule includes a sequence complementary to any RNAhaving TLR2 and/or TLR4 encoding sequence. In another embodiment, anucleic acid molecule may have RNAi activity against TLR2 and/or TLR4RNA, where the nucleic acid molecule includes a sequence complementaryto an RNA having variant TLR2 and/or TLR4 encoding sequence, for exampleother mutant TLR2 and/or TLR4 genes known in the art to be associatedwith the maintenance and/or development of lung disease, disorder orinjury as described herein. In another embodiment, a nucleic acidmolecule disclosed herein includes a nucleotide sequence that caninteract with nucleotide sequence of a TLR2 and/or TLR4 gene and therebymediate silencing of TLR2 and/or TLR4, respectively, gene expression,for example, wherein the nucleic acid molecule mediates regulation ofTLR2 and/or TLR4 gene expression by cellular processes that modulate thechromatin structure or methylation patterns of the TLR2 and/or TLR4 geneand prevent transcription of the TLR2 and/or TLR4 gene.

Antibody Therapy

In some embodiments the inhibitor or therapeutic agent as providedherein comprises and antibody. It should be understood that when theterms “antibody” or “antibodies” are used, this is intended to includeintact antibodies, such as polyclonal antibodies or monoclonalantibodies (mAbs), as well as proteolytic fragments thereof such as theFab or F(ab)₂ fragments. Further included within the scope of theprovided methods and compositions are chimeric antibodies; human andhumanized antibodies; recombinant and engineered antibodies, andfragments thereof. Furthermore, the DNA encoding the variable region ofthe antibody can be inserted into the DNA encoding other antibodies toproduce chimeric antibodies (see, for example, U.S. Pat. No. 4,816,567).Single chain antibodies fall within the scope of the present inventions.Single chain antibodies can be single chain composite polypeptideshaving antigen binding capabilities and comprising amino acid sequenceshomologous or analogous to the variable regions of an immunoglobulinlight and heavy chain (linked VH-VL or single chain Fv (ScFv)). BothV_(H) and V_(L) may copy natural monoclonal antibody sequences or one orboth of the chains may comprise a CDR-FR construct of the type describedin U.S. Pat. No. 5,091,513, the entire contents of which are herebyincorporated herein by reference. The separate polypeptides analogous tothe variable regions of the light and heavy chains are held together bya polypeptide Methods of production of such single chain antibodies,particularly where the DNA encoding the polypeptide structures of theV_(H) and V_(I) chains are known, may be accomplished in accordance withthe methods described, for example, in U.S. Pat. Nos. 4,946,778,5,091,513 and 5,096,815, the entire contents of each of which are herebyincorporated herein by reference.

Additionally, CDR grafting may be performed to alter certain propertiesof the antibody molecule including affinity or specificity. Anon-limiting example of CDR grafting is disclosed in U.S. Pat. No.5,225,539.

Methods of Treatment

Provided herein is a method for treating a lung disorder or injury in amammal in need thereof comprising administering to the mammal at leastone therapeutic agent selected from a TLR2 inhibitor or apharmaceutically acceptable salt or prodrug; in an amount effect totreat the mammal.

In various embodiments the therapeutic agent is selected from the groupconsisting of a small molecule chemical compound; a protein; an antibodyor fragment thereof; a peptide, a peptidomimetic and a nucleic acidmolecule.

Provided herein is a method for treating a lung disorder or injury in amammal in need thereof comprising administering to the mammal at leasttwo therapeutic agents selected from: (i) at least one TLR2 inhibitor ora pharmaceutically acceptable salt or prodrug thereof and (ii) at leastone TLR4 inhibitor or a pharmaceutically acceptable salt or prodrugthereof; in an amount effective to treat the mammal. In some embodimentsthe therapeutic agent is a combined inhibitor by which it is meant asingle agent which is capable of inhibiting the expression and/oractivity of both TLR2 gene or gene products thereof and TLR4 gene orgene products thereof.

In various embodiments each therapeutic agent is independently selectedfrom the group consisting of a small molecule chemical compound; aprotein; an antibody or fragment thereof; a peptide, a peptidomimeticand a nucleic acid molecule.

In one embodiment, nucleic acid molecules may be used to down-regulateor inhibit the expression of TLR2 and TLR4 proteins arising from TLR2and TLR4 haplotype polymorphisms that are associated with a disease orcondition, (e.g. lung disease, disorder or injury as described herein).Analysis of TLR2 and TLR4 genes, or TLR2 and TLR4 protein or RNA levelscan be used to identify subjects with such polymorphisms or thosesubjects who are at risk of developing traits, conditions, or diseasesdescribed herein. These subjects are amenable to treatment, for example,treatment with nucleic acid molecules disclosed herein and any othercomposition useful in treating diseases related to TLR2 and/or TLR4 geneexpression. As such, analysis of TLR2 and/or TLR4 protein or RNA levelscan be used to determine treatment type and the course of therapy intreating a subject. Monitoring of TLR2 and/or TLR4 protein or RNA levelscan be used to predict treatment outcome and to determine the efficacyof compounds and compositions that modulate the level and/or activity ofcertain TLR2 and/or TLR4 proteins associated with a trait, condition, ordisease described herein.

In preferred embodiments the subject being treated is a warm-bloodedanimal and, in particular, mammals including human and non-humanprimates.

Provided are compositions and methods for inhibition of TLR2 and TLR4expression by using small nucleic acid molecules as provided herein,such as short interfering nucleic acid (siNA), double-stranded nucleicacid (dsNA), interfering RNA (RNAi), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules capable of mediating or that mediate RNA interferenceagainst TLR2 and/or TLR4 gene expression. The composition and methodsdisclosed herein are also useful in treating various lung disorders andinjury such as acute respiratory distress syndrome (ARDS), acute lunginjury, pulmonary fibrosis (idiopathic), bleomycin induced pulmonaryfibrosis, mechanical ventilator induced lung injury, chronic obstructivepulmonary disease (COPD), chronic bronchitis, emphysema, bronchiolitisobliterans after lung transplantation and lung transplantation-inducedacute graft dysfunction. The compositions and methods disclosed hereinare also useful in treating or preventing inflammation and/or graftrejection associated with organ transplantation, in particular lungtransplantation, including treatment, prevention or attenuation ofprogression of primary graft failure, ischemia-reperfusion injury,reperfusion injury, reperfusion edema, allograft dysfunction, pulmonaryreimplantation response, bronchiolitis obliterans after lungtransplantation and/or primary graft dysfunction (PGD) after organtransplantation, in particular lung transplantation.

The nucleic acid molecules disclosed herein individually, or incombination or in conjunction with other drugs, can be use forpreventing or treating diseases, traits, conditions and/or disordersassociated with TLR2 and/or TLR4, such as lung disorders or injury andgraft rejection associated with organ transplantation, in particularlung transplantation.

The nucleic acid molecules disclosed herein are able to down-regulatethe expression of TLR2 and/or TLR4 in a sequence specific manner. Thenucleic acid molecules may include a sense strand and an antisensestrand, which includes contiguous nucleotides that are at leastpartially complementary (antisense) to a TLR2 and/or TLR4 mRNA.

In some embodiments, dsRNA specific for TLR2 and/or TLR4 can be used inconjunction with other dsRNA.

Lung disorders and injury can be treated by RNA interference usingnucleic acid molecules as disclosed herein. Exemplary lung disorders andinjuries are disclosed herein. The nucleic acid molecules disclosedherein may inhibit the expression of TLR2 and/or TLR4 in a sequencespecific manner.

Treatment of lung injury can be monitored by determining the level ofPaO2 using suitable techniques known in the art. Treatment can also bemonitored by determining total and differential bronchoalveolar lavage(BAL) counts of different cell populations (e.g. neutrophils,lymphocytes, monocytes, eosinophils, basophils) using suitabletechniques known in the art. Treatment can also be monitored bydetermining the level of TLR2 and/or TLR4 mRNA or the level of TLR2and/or TLR4 protein in the cells of the affected tissue. Treatment canalso be monitored by non-invasive scanning of the affected organ ortissue such as by computer assisted tomography scan, magnetic resonanceelastography scans and other suitable techniques known in the art.

A method for treating or preventing TLR2 associated disease or conditionin a subject or organism may include contacting the subject or organismwith a nucleic acid molecule as provided herein under conditionssuitable to down-regulate the expression of TLR2 gene in the subject ororganism. A method for treating or preventing TLR2 and TLR4 associateddisease or condition in a subject or organism may include contacting thesubject or organism with nucleic acid molecules as provided herein underconditions suitable to down-regulate the expression of TLR2 and TLR4genes in the subject or organism.

A method for treating or preventing lung disease, disorder or injury ina subject or organism may include contacting the subject or organismwith a nucleic acid molecule under conditions suitable to down-regulatethe expression of TLR2 gene in the subject or organism.

A method for treating or preventing lung disease, disorder or injury ina subject or organism may include contacting the subject or organismwith a nucleic acid molecule under conditions suitable to down-regulatethe expression of TLR2 gene and with a nucleic acid molecule underconditions suitable to down-regulate the expression of both, TLR4 gene,in the subject or organism.

A method for treating or preventing one or more lung diseases ordisorders selected from the group consisting of acute respiratorydistress syndrome (ARDS), acute lung injury, pulmonary fibrosis(idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilator induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, emphysema, bronchiolitis obliterans afterlung transplantation and graft rejection associated with organtransplantation, in particular lung transplantation, in a subject ororganism may include contacting the subject or organism with a nucleicacid molecule under conditions suitable to down-regulate the expressionof TLR2 gene in the subject or organism.

A method for treating or preventing one or more lung diseases ordisorders selected from the group consisting of acute respiratorydistress syndrome (ARDS), acute lung injury, pulmonary fibrosis(idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilation induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, emphysema, bronchiolitis obliterans afterlung transplantation and graft rejection associated with organtransplantation, in particular lung transplantation, in a subject ororganism may include contacting the subject or organism with a nucleicacid molecule under conditions suitable to down-regulate the expressionof TLR2 and with a nucleic acid molecule under conditions suitable todown-regulate the expression of TLR4 gene, in the subject or organism.

In various embodiments the provided methods of treating a lung disease,disorder or injury comprise inhibiting the gene Toll-like receptor 2(TLR2) in combination with one or more additional treatment methodsselected from the group consisting of surgery, steroid therapy,non-steroid therapy, antiviral therapy, antifungal therapy,immunosuppressant therapy, anti-infective therapy, anti-hypertensivetherapy and nutritional supplements. In various embodiments the providedmethods of treating a lung disease, disorder or injury, comprisedown-regulating the gene Toll-like receptor 2 (TLR2) in combination withimmunosuppressant therapy.

In various embodiments the provided methods of treating a lung disease,disorder or injury comprise down-regulating the genes Toll-like receptor2 (TLR2) and Toll-like receptor 4 (TLR4) in combination with one or moreadditional treatment methods selected from the group consisting ofsurgery, steroid therapy, non-steroid therapy, antiviral therapy,antifungal therapy, immunosuppressant therapy, anti-infective therapy,anti-hypertensive therapy and nutritional supplements. In variousembodiments the provided methods of treating a lung disease, disorder orinjury comprise down-regulating the Toll-like receptor 2 (TLR2) gene anddown-regulating the Toll-like receptor 4 (TLR4) gene, in combinationwith immunosuppressant therapy.

Lung Disorders and Injury

The methods and compositions disclosed herein are useful in treating asubject experiencing or suffering from or at risk of suffering fromacute respiratory distress syndrome (ARDS), acute lung injury, pulmonaryfibrosis (idiopathic), bleomycin induced pulmonary fibrosis, mechanicalventilator induced lung injury, chronic obstructive pulmonary disease(COPD), chronic bronchitis, emphysema and medical complication of lungtransplantation, including, without being limited to, primary graftfailure, ischemia-reperfusion injury, reperfusion injury, reperfusionedema, allograft dysfunction, pulmonary reimplantation response,bronchiolitis obliterans after lung transplantation and/or primary graftdysfunction (PGD).

Acute Respiratory Distress Syndrome (ARDS)

ARDS is defined as an acute condition characterized by bilateralpulmonary infiltrates and severe hypoxemia in the absence of evidencefor cardiogenic pulmonary edema. Acute respiratory distress syndrome(ARDS) is associated with diffuse alveolar damage (DAD) and lungcapillary endothelial injury. The early phase is described as beingexudative, whereas the later phase is fibroproliferative in character.

Early ARDS is characterized by an increase in the permeability of thealveolar-capillary barrier leading to an influx of fluid into thealveoli. The alveolar-capillary barrier is formed by the microvascularendothelium and the epithelial lining of the alveoli. Hence, a varietyof insults resulting in damage either to the vascular endothelium or tothe alveolar epithelium could result in ARDS. The main site of injurymay be focused on either the vascular endothelium (e.g., sepsis) or thealveolar epithelium (e.g., aspiration of gastric contents).

Injury to the endothelium results in increased capillary permeabilityand the influx of protein-rich fluid into the alveolar space. Injury tothe alveolar lining cells also promotes pulmonary edema formation. Twotypes of alveolar epithelial cells exist. Type I cells, comprising 90%of the alveolar epithelium, are injured easily. Damage to type I cellsallows both increased entry of fluid into the alveoli and decreasedclearance of fluid from the alveolar space. Type II cells have severalimportant functions, including the production of surfactant, iontransport, and proliferation and differentiation into type I cells aftercellular injury. Damage to type II cells results in decreased productionof surfactant with resultant decreased compliance and alveolar collapse.Interference with the normal repair processes in the lung may lead tothe development of fibrosis.

ARDS causes marked increase in intrapulmonary shunt, leading to severehypoxemia. Although high inspired oxygen concentrations are required tomaintain adequate tissue oxygenation and life, additional measures, likelung recruitment with positive end-expiratory pressure (PEEP), is oftenrequired. ARDS is uniformly associated with pulmonary hypertension.Pulmonary artery vasoconstriction likely contributes toventilation-perfusion mismatch and is one of the mechanisms of hypoxemiain ARDS. Normalization of pulmonary artery pressures occurs as thesyndrome resolves. Morbidity is considerable. Patients with ARDS arelikely to have prolonged hospital courses, and they frequently developnosocomial infections, especially ventilator-associated pneumonia. Inaddition, patients often have significant weight loss and muscleweakness and functional impairment may persist for months followinghospital discharge. Most of the deaths in ARDS are attributable tosepsis or multiorgan failure rather than a primary pulmonary cause,although the recent success of mechanical ventilation using smallertidal volumes may suggest a role of lung injury as a direct cause ofdeath.

Acute Lung Injury (ALI)

Acute lung injury (ALI) is a diffuse heterogeneous lung injurycharacterized by hypoxemia, non cardiogenic pulmonary edema, low lungcompliance and widespread capillary leakage. ALI is caused by anystimulus of local or systemic inflammation, principally sepsis.

There are two forms of ALI. Primary ALI is caused by a direct injury tothe lung (e.g., pneumonia). Secondary ALI is caused by an indirectinsult (e.g., pancreatitis). There are two stages—the acute phasecharacterized by disruption of the alveolar-capillary interface, leakageof protein rich fluid into the interstitium and alveolar space, andextensive release of cytokines and migration of neutrophils. A laterreparative phase is characterized by fibroproliferation and remodelingof lung tissue.

The core pathology is disruption of the capillary-endothelial interface:this actually refers to two separate barriers—the endothelium and thebasement membrane of the alveolus. In the acute phase of ALI, there isincreased permeability of this barrier, and protein rich fluid leaks outof the capillaries. There are two types of alveolar epithelial cellsType 1 pneumocytes represent 90% of the cell surface area, and areeasily damaged. Type 2 pneumocytes are more resistant to damage, whichis important as these cells produce surfactant, transport ions andproliferate and differentiate into Type 1 cells.

The damage to the endothelium and the alveolar epithelium results in thecreation of an open interface between the lung and the blood,facilitating the spread of micro-organisms from the lung systemically,stoking up a systemic inflammatory response. Moreover, the injury toepithelial cells handicaps the lung's ability to pump fluid out ofairspaces. Fluid filled airspaces, loss of surfactant, microvascularthrombosis and disorganized repair (which leads to fibrosis) reducesresting lung volumes (decreased compliance), increasingventilation-perfusion mismatch, right to left shunt and the work ofbreathing. In addition, lymphatic drainage of lung units appears to becurtailed—stunned by the acute injury: this contributes to the build upof extravascular fluid.

The patient has low lung volumes, atelectasis, loss of compliance,ventilation-perfusion mismatch (increased deadspace), and right to leftshunt. Clinical features are —severe dyspnea, tachypnea, and resistanthypoxemia.

Prolonged inflammation and destruction of pneumocytes leads tofibroblastic proliferation, hyaline membrane formation and lungfibrosis. This fibrosing alvcolitis may become apparent as early as fivedays after the initial injury. Subsequent recovery may be characterizedby reduced physiologic reserve, and increased susceptibility to furtherlung injuries. Extensive microvascular thrombosis may lead to pulmonaryhypertension, myocardial dysfunction and systemic hypotension.

Pulmonary Fibrosis (Idiopathic)

Idiopathic pulmonary fibrosis (IPF) is an idiopathic interstitialpneumonia that is characterized histopathologically by the presence ofusual interstitial pneumonia. The hallmark pathologic feature of usualinterstitial pneumonia is a heterogeneous, variegated appearance withalternating areas of healthy lung, interstitial inflammation, fibrosis,and honeycomb change. Fibrosis predominates over inflammation.Idiopathic pulmonary fibrosis portends a poor prognosis, and, to date,no proven effective therapies are available for the treatment ofidiopathic pulmonary fibrosis beyond lung transplantation.

The etiology of idiopathic pulmonary fibrosis remains undefined;however, in the current hypothesis regarding the pathogenesis ofidiopathic pulmonary fibrosis (IPF), exposure to an inciting agent (eg,smoke, environmental pollutants, environmental dust, viral infections,gastroesophageal reflux disease, chronic aspiration) in a susceptiblehost may lead to the initial alveolar epithelial damage. This damage maylead to activation of the alveolar epithelial cells, which provokes themigration, proliferation, and activation of mesenchymal cells with theformation of fibroblastic/myofibroblastic foci, leading to theexaggerated accumulation of extracellular matrix with the irreversibledestruction of the lung parenchyma.

Other potential causes of idiopathic pulmonary fibrosis have beenrecognized through the study of familial pulmonary fibrosis. Familialpulmonary fibrosis may represent 20% of all cases of idiopathicpulmonary fibrosis. Genetic mutations in serum surfactant protein C havebeen discovered in some individuals with familial pulmonary fibrosis. Itis believed these mutations in serum surfactant protein C may damagetype II alveolar epithelial cells. Additionally, it has been describedthat mutant telomerase is associated with familial idiopathic pulmonaryfibrosis.

Bleomycin Induced Pulmonary Fibrosis

Bleomycin is a glycopeptide antibiotic that was isolated from a strainof bacterium Streptomyces verticillus. Bleomycin refers to a family ofstructurally related compounds. When used as an anticancer agent, thechemotherapeutical forms are primarily bleomycin A2 and B2. It works bycausing breaks in DNA. The drug is used in the treatment of variety ofmalignancies, including squamous cell carcinoma of the head and neck,cervix, and esophagus; germ cell tumors; testicular cancer; and bothHodgkin and non-Hodgkin lymphoma. Other anti-cancer drugs (such as forexample cyclophosphamide and methotrexate) may cause lung fibrosissimilarly to bleomycin.

A serious complication of bleomycin therapy is pulmonaryfibrosis/interstitial pulmonary fibrosis (also called fibrosingalveolitis) and impaired lung function. Other, less common forms of lunginjury include organizing pneumonia and hypersensitivity pneumonitis.

Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD), also known as chronicobstructive lung disease (COLD), chronic obstructive airway disease(COAD), chronic airflow limitation (CAL) and chronic obstructiverespiratory disease (CORD), refers to chronic bronchitis and emphysema,a pair of commonly co-existing diseases of the lungs in which theairways become narrowed. This leads to a limitation of the flow of airto and from the lungs causing shortness of breath. In clinical practice,COPD is defined by its characteristically low airflow on lung functiontests. In contrast to asthma, this limitation is poorly reversible andusually gets progressively worse over time.

COPD is caused by noxious particles or gas, most commonly from tobaccosmoking, which triggers an abnormal inflammatory response in the lung.The inflammatory response in the larger airways is known as chronicbronchitis, which is diagnosed clinically when people regularly cough upsputum. In the alveoli, the inflammatory response causes destruction ofthe tissues of the lung, a process known as emphysema. The naturalcourse of COPD is characterized by occasional sudden worsenings ofsymptoms called acute exacerbations, most of which are caused byinfections or air pollution.

Both emphysematous destruction and small airway inflammation often arefound in combination in individual patients, leading to the spectrumthat is known as COPD. When emphysema is moderate or severe, loss ofelastic recoil, rather than bronchiolar disease, is the mechanism ofairflow limitation. By contrast, when emphysema is mild, bronchiolarabnormalities are most responsible for the deficit in lung function.Although airflow obstruction in emphysema is often irreversible,bronchoconstriction due to inflammation accounts for a limited amount ofreversibility.

Pathological changes in chronic obstructive pulmonary disease (COPD)occur in the large (central) airways, the small (peripheral)bronchioles, and the lung parenchyma. The pathogenic mechanisms are notclear but most likely involve diverse mechanisms. The increased numberof activated polymorphonuclear leukocytes and macrophages releaseelastases in a manner that cannot be counteracted effectively byantiproteases, resulting in lung destruction. The primary offender hasbeen human leukocyte elastase, with a possible synergistic rolesuggested for proteinase 3 and macrophage-derived matrix proteinases,cysteine proteinases, and a plasminogen activator. Additionally,increased oxidative stress caused by free radicals in cigarette smoke,the oxidants released by phagocytes, and polymorphonuclear leukocytesall may lead to apoptosis or necrosis of exposed cells. Acceleratedaging and autoimmune mechanisms have also been proposed as having rolesin the pathogenesis of COPD.

Chronic Bronchitis

Chronic bronchitis is a chronic inflammation of the bronchi (medium-sizeairways) in the lungs. It is generally considered one of the two formsof chronic obstructive pulmonary disease (COPD). It is definedclinically as a persistent cough that produces sputum and mucus, for atleast three months in two consecutive years. Mucous gland enlargement isthe histologic hallmark of chronic bronchitis. The structural changesdescribed in the airways include atrophy, focal squamous metaplasia,ciliary abnormalities, variable amounts of airway smooth musclehyperplasia, inflammation, and bronchial wall thickening Neutrophiliadevelops in the airway lumen, and neutrophilic infiltrates accumulate inthe submucosa. The respiratory bronchioles display a mononuclearinflammatory process, lumen occlusion by mucous plugging, goblet cellmetaplasia, smooth muscle hyperplasia, and distortion due to fibrosis.These changes, combined with loss of supporting alveolar attachments,cause airflow limitation by allowing airway walls to deform and narrowthe airway lumen.

Emphysema

Emphysema is a long-term, progressive disease of the lungs thatprimarily causes shortness of breath. In people with emphysema, thetissues necessary to support the physical shape and function of thelungs are destroyed. It is included in a group of COPD. Emphysema iscalled an obstructive lung disease because the destruction of lungtissue around smaller sacs, called alveoli, makes these air sacs unableto hold their functional shape upon exhalation. It is often caused bysmoking or long-term exposure to air pollution.

Emphysema has 3 morphologic patterns. The first type, centriacinaremphysema, is characterized by focal destruction limited to therespiratory bronchioles and the central portions of acinus. This form ofemphysema is associated with cigarette smoking and is most severe in theupper lobes. The second type, panacinar emphysema, involves the entirealveolus distal to the terminal bronchiole. The panacinar type is mostsevere in the lower lung zones and generally develops in patients withhomozygous alpha1-antitrypsin (AAT) deficiency. The third type, distalacinar emphysema or paraseptal emphysema, is the least common form andinvolves distal airway structures, alveolar ducts, and sacs. This formof emphysema is localized to fibrous septa or to the pleura and leads toformation of bullae. The apical bullae may cause pneumothorax.Paraseptal emphysema is not associated with airflow obstruction.

Lung Transplantation and its Complications

The term “lung transplantation” is meant to encompass a surgicalprocedure in which a patient's diseased lungs are partially or totallyreplaced by lungs which come from a donor. Although a xenotransplant canbe contemplated in certain situations, an allotransplant is usuallypreferable.

Lung transplantation has become a treatment of choice for patients withadvanced/end-stage lung diseases. Indications for lung transplantationinclude chronic obstructive pulmonary disease (COPD), pulmonaryhypertension, cystic fibrosis, idiopathic pulmonary fibrosis, andEisenmenger syndrome. Typically, four different surgical techniques areused: single-lung transplantation, bilateral sequential transplantation,combined heart-lung transplantation, and lobar transplantation, with themajority of organs obtained from deceased donors. Within last decades,donor management, organ preservation, immunosuppressive regimens andcontrol of infectious complications have been substantially improved andthe operative techniques of transplantation procedures have beendeveloped. Nonetheless, primary graft dysfunction (PGD) affects anestimated 10 to 25% of lung transplants and is the leading cause ofearly post-transplantation morbidity and mortality for lung recipients(Lee J C and Christie J D. 2009. Proc Am Thorac Soc, vol. 6: 39-46). PGDmanifests as an acute lung injury defined by diffuse infiltrates onchest x-ray and abnormal oxygenation. There, there is some evidence tosuggest a relationship between reperfusion injury, acute rejection, andthe subsequent development of chronic graft dysfunction. Chronicrejection, known as obliterative bronchiolitis/bronchiolitis obliteranssyndrome (BOS), is the key reason why the five year survival is only50%, which is significantly worse than most other solid organtransplants. Investigators have recently demonstrated that PGD increasesthe risk of the development of BOS independent of other risk factors,and the severity of PGD is directly associated with increased risk forBOS (Daud S A, Yusen R D et al. 2007 Am J Respir Crit Care Med. 2007;175(5):507-513).

Bronchiolitis Obliterans after Lung Transplantation

Bronchiolitis obliterans, and its clinical correlate bronchiolitisobliterans syndrome, affect up to 50-60% of patients who survive 5 yearsafter transplantation. In most patients, bronchiolitis obliterans is aprogressive process that responds poorly to augmented immunosuppression,and it accounts for more than 30% of all deaths occurring after thethird postoperative year. Survival at 5 years after the onset ofbronchiolitis obliterans is only 30-40%, and survival at 5 years aftertransplantation is 20-40% lower in patients with than in patientswithout bronchiolitis obliterans.

The diagnosis of bronchiolitis obliterans is based on histology, buthistologic proof is often difficult to obtain using transbronchial lungbiopsies. Therefore, in 1993, a committee sponsored by the InternationalSociety for Heart and Lung Transplantation (ISHLT) proposed a clinicaldescription of bronchiolitis obliterans, termed BOS, which is based onchanges in FEV₁. For each patient, a stable post-transplant baselineFEV₁ is defined as BOS stage 0; in patients who experience a decrease inFEV₁, progressive stages of BOS, from 1 to 3, are defined according tothe magnitude of the decrease. Although this classification system hasbeen adopted by transplant centers worldwide as a useful descriptor ofchronic allograft dysfunction, concern has been raised regarding itsability to detect small changes in pulmonary function. This concernrecently led to formulation of a revised classification system for BOS,which includes a new “potential-BOS” stage (BOS 0-p) defined as adecrease in midexpiratory flow rates (FE₂₅₋₇₅) and/or FEV₁. Therationale for including FEF₂₅₋₇₅ comes from studies in heart-lung andbilateral lung recipients, which showed that this variable deterioratesbefore FEV₁ at the onset of BOS. The new BOS 0-p stage is meant to alertthe physician and to indicate the need for close functional monitoringand for in-depth assessment using surrogate markers for BOS. However,the usefulness of stage BOS 0-p in recipients of single lungs, inparticular those with emphysema, still needs to be established.

The histopathological features of bronchiolitis obliterans suggest thatinjury and inflammation of epithelial cells and subepithelial structuresof small airways lead to excessive fibroproliferation due to ineffectiveepithelial regeneration and aberrant tissue repair. In parallel with theconcept of “injury response” that has been proposed to explain chronicdysfunction of other organ allografts, airway injury may occur viaalloimmune-dependent and -independent mechanisms acting alone or incombination. The evolving concept is that bronchiolitis obliteransrepresents a “final common pathway” lesion, in which various insults canlead to a similar histological and clinical result. Yet the rarity ofthis syndrome in untransplanted individuals suggests thatalloimmune-dependent mechanisms usually play a pivotal role.

Delivery of Nucleic Acid Molecules and Pharmaceutical Formulations

Nucleic acid molecules may be adapted for use to prevent or treat lungdiseases, injuries, traits, conditions and/or disorders, alone or incombination with other therapies. A nucleic acid molecule may include adelivery vehicle, including liposomes, for administration to a subject,carriers and diluents and their salts, and/or can be present inpharmaceutically acceptable formulations.

Nucleic acid molecules disclosed herein may be delivered or administeredas the compound per se (i.e. naked nucleic acid molecule) or aspharmaceutically acceptable salt and may be delivered or administeredalone or as an active ingredient in combination with one or morepharmaceutically acceptable carrier, solvent, diluent, excipient,adjuvant and/or vehicle. In some embodiments, nucleic acid moleculesdisclosed herein are delivered to the target tissue by directapplication of the naked molecules prepared with a carrier or a diluent.

The term “naked nucleic acid molecule” refers to nucleic acid moleculesthat are free from any delivery vehicle that acts to assist, promote orfacilitate entry into the cell, including e.g. viral vectors, viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. For example, siRNA in PBS is “nakedsiRNA”.

Nucleic acid molecules may be delivered or administered to a subject bydirect application of the nucleic acid molecules with a carrier ordiluent or any other delivery vehicle that acts to assist, promote orfacilitate entry into a cell, including e.g. viral vectors, viralsequences, viral particular, liposome formulations, lipofectin orprecipitating agents and the like. Polypeptides that facilitateintroduction of nucleic acid into a desired subject are described in USApplication Publication No. 20070155658 (e.g., a melamine derivativesuch as 2,4,6-Triguanidino Traizine and 2,4,6-Tramidosarcocyl Melamine,a polyarginine polypeptide, and a polypeptide including alternatingglutamine and asparagine residues).

Methods for the delivery of nucleic acid molecules are described inAkhtar et al., Trends Cell Bio., 2: 139 (1992); Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer etal., Mol. Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb.Exp. Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser.,752: 184-192 (2000); U.S. Pat. Nos. 6,395,713; 6,235,310; 5,225,182;5,169,383; 5,167,616; 4,959217; 4,925,678; 4,487,603; and 4,486,194 andSullivan et al., PCT WO 94/02595; PCT WO 00/03683 and PCT WO 02/08754;and U.S. Patent Application Publication No. 2003077829. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see e.g., Gonzalez et al., Bioconjugatc Chem.,10: 1068-1074 (1999); Wang et al., International PCT publication Nos. WO03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCAmicrospheres (see for example U.S. Pat. No. 6,447,796 and U.S.Application Publication No. 2002130430), biodegradable nanocapsules, andbioadhesive microspheres, or by proteinaceous vectors (O'Hare andNormand, International PCT Publication No. WO 00/53722). Alternatively,the nucleic acid composition/combination is locally delivered by directinjection, oral instillation, inhalation or by use of an infusion pump.Direct injection of the nucleic acid molecules as provided herein,whether e.g. intratracheal, subcutaneous, intramuscular, or intradermal,can take place using standard needle and syringe methodologies, or byneedle-free technologies such as those described in Conry et al., Clin.Cancer Res., 5: 2330-2337 (1999) and Barry et al., International PCTPublication No. WO 99/31262. The molecules provided herein can be usedas pharmaceutical agents. Pharmaceutical agents prevent, modulate theoccurrence, or treat (alleviate a symptom to some extent, preferably allof the symptoms) of a disease state in a subject.

Nucleic acid molecules may be complexed with cationic lipids, packagedwithin liposomes, or otherwise delivered to target cells or tissues. Thenucleic acid or nucleic acid complexes can be locally administered torelevant tissues ex vivo, or in vivo through direct dermal application,transdermal application, or injection, with or without theirincorporation in biopolymers.

Delivery systems include surface-modified liposomes containing poly(ethylene glycol) lipids (PEG-modified, or long-circulating liposomes orstealth liposomes). These formulations offer a method for increasing theaccumulation of drugs in target tissues. This class of drug carriersresists opsonization and elimination by the mononuclear phagocyticsystem (MPS or RES), thereby enabling longer blood circulation times andenhanced tissue exposure for the encapsulated drug (Lasic et al. Chem.Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43,1005-1011).

Nucleic acid molecules may be formulated or complexed withpolyethylenimine (e.g., linear or branched PEI) and/or polyethyleniminederivatives, including for examplepolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneiminepolyethylene-glycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives, grafted PEIs such as galactose PEI,cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI(PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPAPharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugatc Chem., 14,840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choiet al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al.,1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002,Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of GeneMedicine Preprint, 1, 1-18; Godbey et al., 1999, PNAS USA, 96,5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60,149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645;Sagara, U.S. Pat. No. 6,586,524 and United States Patent ApplicationPublication No. 20030077829).

Nucleic acid molecules may be complexed with membrane disruptive agentssuch as those described in U.S. Patent Application Publication No.20010007666. The membrane disruptive agent or agents and the nucleicacid molecule may also be complexed with a cationic lipid or helperlipid molecule, such as those lipids described in U.S. Pat. No.6,235,310.

The nucleic acid molecules may be delivered or administered via apulmonary delivery, such as by inhalation of an aerosol or spray driedformulation administered by an inhalation device or nebulizer, providingrapid local uptake of the nucleic acid molecules into relevant pulmonarytissues. Solid particulate compositions containing respirable dryparticles of micronized nucleic acid compositions can be prepared bygrinding dried or lyophilized nucleic acid compositions, and thenpassing the micronized composition through, for example, a 400 meshscreen to break up or separate out large agglomerates. A solidparticulate composition comprising the nucleic acid compositionsprovided herein can optionally contain a dispersant which serves tofacilitate the formation of an aerosol as well as other therapeuticcompounds. A suitable dispersant is lactose, which can be blended withthe nucleic acid compound in any suitable ratio, such as a 1 to 1 ratioby weight.

Aerosols of liquid particles may include a nucleic acid moleculesdisclosed herein and can be produced by any suitable means, such as witha nebulizer (see e.g., U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers include the active ingredient(s) in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, e.g., sodium chloride or other suitable salts. Optionaladditives include preservatives if the formulation is not preparedsterile, e.g., methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. The aerosols of solid particles including the activecomposition and surfactant can likewise be produced with any solidparticulate aerosol generator. Aerosol generators for administeringsolid particulate therapeutics to a subject produce particles, which arerespirable, as explained above, and generate a volume of aerosolcontaining a predetermined metered dose of a therapeutic composition ata rate suitable for human administration. One illustrative type of solidparticulate aerosol generator is an insufflator. Suitable formulationsfor administration by insufflation include finely comminuted powders,which can be delivered by means of an insufflator. In the insufflator,the powder, e.g., a metered dose thereof effective to carry out thetreatments described herein, is contained in capsules or cartridges,typically made of gelatin or plastic, which are either pierced or openedin situ and the powder delivered by air drawn through the device uponinhalation or by means of a manually-operated pump. The powder employedin the insufflator consists either solely of the active ingredient or ofa powder blend comprising the active ingredient(s), a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient(s) typically includes from 0.1 to 100 w/w of the formulation.A second type of illustrative aerosol generator includes a metered doseinhaler. Metered dose inhalers are pressurized aerosol dispensers,typically containing a suspension or solution formulation of the activeingredient in a liquefied propellant. During use these devices dischargethe formulation through a valve adapted to deliver a metered volume toproduce a fine particle spray containing the active ingredient(s).Suitable propellants include certain chlorofluorocarbon compounds, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, e.g., US PatentApplication Publication No. 20040037780, and U.S. Pat. Nos. 6,592,904;6,582,728; 6,565,885.

Delivery systems may include, for example, aqueous and nonaqueous gels,creams, multiple emulsions, microemulsions, liposomes, ointments,aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon basesand powders, and can contain excipients such as solubilizers, permeationenhancers (e.g., fatty acids, fatty acid esters, fatty alcohols andamino acids), and hydrophilic polymers (e.g., polycarbophil andpolyvinylpyrolidone). In one embodiment, the pharmaceutically acceptablecarrier is a liposome or a transdermal enhancer. Examples of liposomeswhich can be used in accordance with the compositions and methodsprovided herein include the following: (1) CellFectin, 1:1.5 (M/M)liposome formulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidyl-ethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA, the neutral lipid DOPE(GIBCO BRL) and Di-Alkylated Amino Acid (DiLA2).

Nucleic acid molecules may include a bioconjugate, for example a nucleicacid conjugate as described in Vargeese et al., U.S. Ser. No.10/427,160; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat.No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S.Pat. No. 5,138,045.

Expression of Nucleic Acid Molecules

Compositions, methods and kits disclosed herein may include anexpression vector that includes a nucleic acid sequence encoding atleast one nucleic acid molecule of such as provided herein in a mannerthat allows expression of the nucleic acid molecule. Methods ofintroducing nucleic acid molecules or one or more vectors capable ofexpressing the strands of dsRNA into the environment of the cell willdepend on the type of cell and the make up of its environment. Thenucleic acid molecule or the vector construct may be directly introducedinto the cell (i.e., intracellularly); or introduced extracellularlyinto a cavity, interstitial space, into the circulation of an organism,introduced orally, or may be introduced by bathing an organism or a cellin a solution containing dsRNA. The cell is preferably a mammalian cell;more preferably a human cell. The nucleic acid molecule of theexpression vector can include a sense region and an antisense region.The antisense region can include a sequence complementary to a RNA orDNA sequence encoding a gene selected from a TLR2 gene and a TLR4 gene;and the sense region can include a sequence complementary to theantisense region. The nucleic acid molecule can include two distinctstrands having complementary sense and antisense regions. The nucleicacid molecule can include a single strand having complementary sense andantisense regions.

Nucleic acid molecules that interact with target RNA molecules anddown-regulate gene encoding target RNA molecules (e.g., target RNAmolecules referred to by Genbank Accession numbers herein) may beexpressed from transcription units inserted into DNA or RNA vectors.Recombinant vectors can be DNA plasmids or viral vectors. Nucleic acidmolecule expressing viral vectors can be constructed based on, but notlimited to, adeno-associated virus, retrovirus, adenovirus, oralphavirus. The recombinant vectors capable of expressing the nucleicacid molecules can be delivered as described herein, and persist intarget cells. Alternatively, viral vectors can be used that provide fortransient expression of nucleic acid molecules. Such vectors can berepeatedly administered as necessary. Once expressed, the nucleic acidmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of nucleic acid molecule expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by direct administration to the lung, e.g. byintratracheal injection, by administration to target cells ex-plantedfrom a subject followed by reintroduction into the subject, or by anyother means that would allow for introduction into the desired targetcell.

Expression vectors may include a nucleic acid sequence encoding at leastone nucleic acid molecule disclosed herein, in a manner which allowsexpression of the nucleic acid molecule. For example, the expressionvector may encode one or both strands of a nucleic acid duplex, or asingle self-complementary strand that self hybridizes into a nucleicacid duplex. The nucleic acid sequences encoding nucleic acid moleculescan be operably linked in a manner that allows expression of the nucleicacid molecule. Non-limiting examples of such expression vectors aredescribed in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishiand Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, NatureBiotechnology, 19, 500; and Novina et al., 2002, Nature Medicine,advance online publication doi:10.1038/nm725. Expression vectors mayalso be included in a mammalian (e.g., human) cell.

An expression vector may include a nucleic acid sequence encoding two ormore nucleic acid molecules, which can be the same or different.Expression vectors may include a sequence for a nucleic acid moleculecomplementary to a nucleic acid molecule referred to by a GenbankAccession number NM_003264.3 (TLR2), NR_024169.1 (TLR4), NM_138554.3(TLR4) or NR_024168.1 (TLR4).

An expression vector may include one or more of the following: a) atranscription initiation region (e.g., eukaryotic pol I, II or IIIinitiation region); b) a transcription termination region (e.g.,eukaryotic pol I, II or III termination region); c) an intron and d) anucleic acid sequence encoding at least one of the nucleic acidmolecules, wherein said sequence is operably linked to the initiationregion and the termination region in a manner that allows expressionand/or delivery of the nucleic acid molecule. The vector can optionallyinclude an open reading frame (ORF) for a protein operably linked on the5′-side or the 3′-side of the sequence encoding the nucleic acidmolecule; and/or an intron (intervening sequences).

Transcription of the nucleic acid molecule sequences can be driven froma promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II(pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters are expressed at high levels in all cells; the levelsof a given pol II promoter in a given cell type depends on the nature ofthe gene regulatory sequences (enhancers, silencers, etc.) presentnearby. Prokaryotic RNA polymerase promoters are also used, providingthat the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy-Stein and Moss, 1990, PNAS USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabot et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, PNAS USA, 89, 10802-6; Chen et al., 1992, Nucleic AcidsRes., 20, 4581-9; Yu et al., 1993, PNAS USA, 90, 6340-4; L'Huillier etal., 1992, EMBO J., 11, 4411-8; Lisziewicz et al., 1993, PNAS USA, 90,8000-4; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger &Cech, 1993, Science, 262, 1566). More specifically, transcription unitssuch as the ones derived from genes encoding U6 small nuclear RNA(snRNA), transfer RNA (tRNA) and adenovirus VA RNA are useful ingenerating high concentrations of desired RNA molecules such as siNA incells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra;Noonberg et al., 1994, Nucleic Acid Res., 22, 2830; Noonberg et al.,U.S. Pat. No. 5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelmanet al., International PCT Publication No. WO 96/18736). The abovenucleic acid transcription units can be incorporated into a variety ofvectors for introduction into mammalian cells, including but notrestricted to, plasmid DNA vectors, viral DNA vectors (such asadenovirus or adeno-associated virus vectors), or viral RNA vectors(such as retroviral or alphavirus vectors) (see Couture and Stinchcomb,1996 supra).

A nucleic acid molecule may be expressed within cells from eukaryoticpromoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarryand Lindquist, 1986, PNAS USA 83, 399; Scanlon et al., 1991, PNAS USA,88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15;Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991,J. Virol., 65, 5531-4; Ojwang et al., 1992, PNAS USA, 89, 10802-6; Chenet al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990Science, 247, 1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23,2259; Good et al., 1997, Gene Therapy, 4, 45). Those skilled in the artrealize that any nucleic acid can be expressed in eukaryotic cells fromthe appropriate DNA/RNA vector. The activity of such nucleic acids canbe augmented by their release from the primary transcript by a enzymaticnucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCTWO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27, 15-6;Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al.,1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J. Biol.Chem., 269, 25856).

A viral construct packaged into a viral particle would accomplish bothefficient introduction of an expression construct into the cell andtranscription of dsRNA construct encoded by the expression construct.

Methods for oral introduction include direct mixing of RNA with food ofthe organism, as well as engineered approaches in which a species thatis used as food is engineered to express an RNA, then fed to theorganism to be affected. Physical methods may be employed to introduce anucleic acid molecule solution into the cell. Physical methods ofintroducing nucleic acids include injection of a solution containing thenucleic acid molecule, bombardment by particles covered by the nucleicacid molecule, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the nucleic acidmolecule.

Other methods known in the art for introducing nucleic acids to cellsmay be used, such as lipid-mediated carrier transport, chemical mediatedtransport, such as calcium phosphate, and the like. Thus the nucleicacid molecules may be introduced along with components that perform oneor more of the following activities: enhance RNA uptake by the cell,promote annealing of the duplex strands, stabilize the annealed strands,or otherwise increase inhibition of the target gene.

Nucleic Acid Formulations

The nucleic acid molecules or the vector construct can be introducedinto the cell using suitable formulations, e.g. a lipid formulation suchas in Lipofectamine™ 2000 (Invitrogen, CA, USA), vitamin A coupledliposomes (Sato et al. Nat Biotechnol 2008; 26:431-442, PCT PatentPublication No. WO 2006/068232). Lipid formulations can also beadministered to animals such as by intravenous, intramuscular, orintraperitoneal injection, or intratracheal injection, or orally or byinhalation or other methods as are known in the art. When theformulation is suitable for administration into animals such as mammalsand more specifically humans, the formulation is also pharmaceuticallyacceptable. Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used. In some instances, it may bepreferable to formulate nucleic acid molecules, e.g. dsRNA, in a bufferor saline solution and directly inject the formulated dsRNA into thetarget organ or into target cells, as in studies with oocytes. Thedirect injection of dsRNA duplexes may also be done. For suitablemethods of introducing dsRNA see for example U.S. published patentapplication No. 2004/0203145, 20070265220, which are incorporated hereinby reference.

Pharmaceutically acceptable formulations for treating lung disorders orinjury are known and can be used for administration of the therapeuticcombinations disclosed herein. In some instances, the therapeuticcompositions disclosed herein may be formulated for intravenousadministration for systemic delivery, or as aerosols, for example forintranasal administration, or as nasal drops, for example for intranasalinstillation, or as suitable for intratracheal instillation.

Polymeric nanocapsules or microcapsules facilitate transport and releaseof the encapsulated or bound nucleic acid molecule, e.g. dsRNA, into thecell. They include polymeric and monomeric materials, e.g. especiallyincluding polybutylcyanoacrylate. A summary of materials and fabricationmethods has been published (see Kreuter, 1991). The polymeric materialswhich are formed from monomeric and/or oligomeric precursors in thepolymerization/nanoparticle generation step, are per se known from theprior art, as are the molecular weights and molecular weightdistribution of the polymeric material which a person skilled in thefield of manufacturing nanoparticles may suitably select in accordancewith the usual skill.

Nucleic acid molecules may be formulated as a microemulsion. Amicroemulsion is a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution.Typically microemulsions are prepared by first dispersing an oil in anaqueous surfactant solution and then adding a sufficient amount of a 4thcomponent, generally an intermediate chain-length alcohol to form atransparent system.

Surfactants that may be used in the preparation of microemulsionsinclude, but are not limited to, ionic surfactants, non-ionicsurfactants, Brij 96, polyoxyethylene olcyl ethers, polyglycerol fattyacid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate(MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate(PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate(MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate(DA0750), alone or in combination with cosurfactants. The cosurfactant,usually a short-chain alcohol such as ethanol, 1-propanol, and1-butanol, serves to increase the interfacial fluidity by penetratinginto the surfactant film and consequently creating a disordered filmbecause of the void space generated among surfactant molecules.

Water Soluble Crosslinked Polymers

Delivery formulations can include water soluble degradable crosslinkedpolymers that include one or more degradable crosslinking lipid moiety,one or more PEI moiety, and/or one or more mPEG (methyl ether derivativeof PEG (methoxypoly (ethylene glycol)).

The degradable crosslinking lipid moiety may be reacted with apolyethyleneimine (PEI) as shown in Scheme A below:

The reaction illustrated in Scheme A may be carried out by intermixingthe PEI and the diacrylate (I) in a mutual solvent such as ethanol,methanol or dichloromethane with stirring, preferably at roomtemperature for several hours, then evaporating the solvent to recoverthe resulting polymer. While not wishing to be bound to any particulartheory, it is believed that the reaction between the PEI and diacrylate(I) involves a Michael reaction between one or more amines of the PETwith double bond(s) of the diacrylate (see J. March, Advanced OrganicChemistry 3rd Ed., pp. 711-712 (1985)). The diacrylate shown in Scheme Amay be prepared in the manner as described in U.S. application Ser. No.11/216,986 (US Publication No. 2006/0258751).

The molecular weight of the PEI is preferably in the range of about 200to 25,000 Daltons more preferably 400 to 5,000 Daltons, yet morepreferably 600 to 2,000 Daltons. PEI may be either branched or linear.

The molar ratio of PEI to diacrylate is preferably in the range of about1:2 to about 1:20. The weight average molecular weight of the cationiclipopolymer may be in the range of about 500 Daltons to about 1,000,000Daltons preferably in the range of about 2,000 Daltons to about 200,000Daltons. Molecular weights may be determined by size exclusionchromatography using PEG standards or by agarose gel electrophoresis.

The cationic lipopolymer is preferably degradable, more preferablybiodegradable, e.g., degradable by a mechanism selected from the groupconsisting of hydrolysis, enzyme cleavage, reduction, photo-cleavage,and sonication. While not wishing to be bound to any particular theory,it is believed that degradation of the cationic lipopolymer of formula(II) within the cell proceeds by enzymatic cleavage and/or hydrolysis ofthe ester linkages.

Synthesis may be carried out by reacting the degradable lipid moietywith the PEI moiety as described above. Then the mPEG (methyl etherderivative of PEG (methoxypoly (ethylene glycol)), is added to form thedegradable crosslinked polymer. In preferred embodiments, the reactionis carried out at room temperature. The reaction products may beisolated by any means known in the art including chromatographictechniques. In a preferred embodiment, the reaction product may beremoved by precipitation followed by centrifugation.

Dose and Dosage Units

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular recipient andregion thereof to be treated, the particular nucleic acid and deliverymethod used, the therapeutic or diagnostic use contemplated, and theform of the formulation, for example, suspension, emulsion, micelle orliposome, as will be readily apparent to those skilled in the art.Typically, dosage is administered at lower levels and increased untilthe desired effect is achieved.

When lipids are used to deliver the nucleic acid, the amount of lipidcompound that is administered can vary and generally depends upon theamount of nucleic acid being administered. For example, the weight ratioof lipid compound to nucleic acid is preferably from about 1:1 to about30:1, with a weight ratio of about 5:1 to about 10:1 being morepreferred.

A suitable dosage unit of nucleic acid molecules may be in the range ofabout 0.001 to 20-100 milligrams per kilogram body weight of therecipient per day, or in the range of 0.01 to 20 milligrams per kilogrambody weight per day, or in the range of 0.01 to 10 milligrams perkilogram body weight per day, or in the range of 0.1 to 5 milligrams perkilogram body weight per day, or in the range of 0.1 to 2.5 milligramsper kilogram body weight per day, in a regimen of a single dose or aseries of doses given at short (e.g. 1-5 minute) or long (e.g. severalhours) intervals.

In certain embodiment a suitable dosage unit of nucleic acid moleculesmay be in the range of about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05mg, 0.0.6 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg,0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg,1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg,2.3 mg, 2.4 mg, 2.5 mg, 2.6 mg, 2.7 mg, 2.8 mg, 2.9 mg, 3.0 mg, 3.1 mg,3.2 mg, 3.3 mg, 3.4 mg, 3.5 mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4.0 mg,4.1 mg, 4.2 mg, 4.3 mg, 4.4 mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg,5.0 mg, 5.1 mg, 5.2 mg, 5.3 mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg,5.9 mg, 6.0 mg, 6.1 mg, 6.2 mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg,6.8 mg, 6.9 mg, 7.0 mg, 7.1 mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg,7.7 mg, 7.8 mg, 7.9 mg, 8.0 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg,8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9.0 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg,9.5 mg, 9.6 mg, 9.7 mg, 9.8 mg, 9.9 mg, 10.0 mg, 10.1 mg, 10.2 mg, 10.3mg, 10.4 mg, 10.5 mg, 10.6 mg, 10.7 mg, 10.8 mg, 10.9 mg, 11.0 mg, 11.1mg, 11.2 mg, 11.3 mg, 11.4 mg, 11.5 mg, 11.6 mg, 11.7 mg, 11.8 mg, 11.9mg, 12.0 mg, 12.1 mg, 12.2 mg, 12.3 mg, 12.4 mg, 12.5 mg, 12.6 mg, 12.7mg, 12.8 mg, 12.9 mg, 13.0 mg, 13.1 mg, 13.2 mg, 13.3 mg, 13.4 mg, 13.5mg, 13.6 mg, 13.7 mg, 13.8 mg, 13.9 mg, 14.0 mg, 14.1 mg, 14.2 mg, 14.3mg, 14.4 mg, 14.5 mg, 14.6 mg, 14.7 mg, 14.8 mg, 14.9 mg, 15.0 mg, 15.1mg, 15.2 mg, 15.3 mg, 15.4 mg, 15.5 mg, 15.6 mg, 15.7 mg, 15.8 mg, 15.9mg, 16.0 mg, 16.1 mg, 16.2 mg, 16.3 mg, 16.4 mg, 16.5 mg, 16.6 mg, 16.7mg, 16.8 mg, 16.9 mg, 17.0 mg, 17.1 mg, 17.2 mg, 17.3 mg, 17.4 mg, 17.5mg, 17.6 mg, 17.7 mg, 17.8 mg, 17.9 mg, 18.0 mg, 18.1 mg, 18.2 mg, 18.3mg, 18.4 mg, 18.5 mg, 18.6 mg, 18.7 mg, 18.8 mg, 18.9 mg, 19.0 mg, 19.1mg, 19.2 mg, 19.3 mg, 19.4 mg, 19.5 mg, 19.6 mg, 19.7 mg, 19.8 mg, 19.9mg, 20.0 mg per kilogram body weight of the recipient per day. in aregimen of a single dose or a series of doses given at short (e.g. 1-5minute) or long (e.g. several hours) intervals.

Suitable amounts of nucleic acid molecules may be introduced and theseamounts can be empirically determined using standard methods. Effectiveconcentrations of individual nucleic acid molecule species in theenvironment of a cell may be about 1 femtomolar, about 50 femtomolar,100 femtomolar, 1 picomolar, 1.5 picomolar, 2.5 picomolar, 5 picomolar,10 picomolar, 25 picomolar, 50 picomolar, 100 picomolar, 500 picomolar,1 nanomolar, 2.5 nanomolar, 5 nanomolar, 10 nanomolar, 25 nanomolar, 50nanomolar, 100 nanomolar, 500 nanomolar, 1 micromolar, 2.5 micromolar, 5micromolar, 10 micromolar, 100 micromolar or more.

Dosage of each therapeutic agent may be independently from about 0.01 μgto about 1 g per kg of body weight (e.g., 0.1 μg, 0.25 μg, 0.5 μg, 0.75μg, 1 μg, 2.5 μg, 5 μg, 10 μg, 25 μg, 50 μg, 100 μg, 250 μg, 500 μg, 1mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, or 500 mg, or 1g, per kg of body weight).

In certain embodiments dosage levels of the order of from about 0.1 mgto about 140 mg per kilogram of body weight per day are useful in thetreatment of the above-indicated conditions (about 0.5 mg to about 7 gper subject per day). The amount of active ingredient that can becombined with the carrier materials to produce a single dosage formvaries depending upon the host treated and the particular mode ofadministration. Dosage unit forms generally contain between from about 1mg to about 500 mg of an active ingredient.

In certain embodiments, the double-stranded RNA compound is present inthe composition in a dose level of about 0.05 mg, 0.06 mg, 0.07 mg, 0.08mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8mg, 0.9 mg, 1.0 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7mg, 1.8 mg, 1.9 mg, 2.0 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6mg, 2.7 mg, 2.8 mg, 2.9 mg, 3.0 mg, 3.1 mg, 3.2 mg, 3.3 mg, 3.4 mg, 3.5mg, 3.6 mg, 3.7 mg, 3.8 mg, 3.9 mg, 4.0 mg, 4.1 mg, 4.2 mg, 4.3 mg, 4.4mg, 4.5 mg, 4.6 mg, 4.7 mg, 4.8 mg, 4.9 mg, 5.0 mg, 5.1 mg, 5.2 mg, 5.3mg, 5.4 mg, 5.5 mg, 5.6 mg, 5.7 mg, 5.8 mg, 5.9 mg, 6.0 mg, 6.1 mg, 6.2mg, 6.3 mg, 6.4 mg, 6.5 mg, 6.6 mg, 6.7 mg, 6.8 mg, 6.9 mg, 7.0 mg, 7.1mg, 7.2 mg, 7.3 mg, 7.4 mg, 7.5 mg, 7.6 mg, 7.7 mg, 7.8 mg, 7.9 mg, 8.0mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9mg, 9.0 mg, 9.1 mg, 9.2 mg, 9.3 mg, 9.4 mg, 9.5 mg, 9.6 mg, 9.7 mg, 9.8mg, 9.9 mg, or 10.0 mg per dose form.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, frequency of treatment, route of administration, andrate of excretion, drug combination and the severity of the particulardisease undergoing therapy.

Regimens for continuing therapy, including dose and frequency may beguided by the initial response and clinical judgment.

The pulmonary route of administration is preferred, such as byintratracheal instillation, inhalation of an aerosol formulation,although other routes, may be required in specific administration, asfor example to the mucous membranes of the nose, throat, bronchialtissues or lungs. Transdermal route of administration may also be used,including active systems where delivery is driven by microneedles orenergy applied via ultrasound or lasers.

The therapeutic compositions disclosed herein are preferablyadministered into the lung of a subject suffering from lung injury,disorder, disease or who has undergone lung transplantation, byinhalation of an aerosol containing the composition/combination, byintranasal or intratracheal instillation or by inhalation viaventilation machine (e.g. for administration to an unconscious patient).In some embodiments the oligouncleotide compositions disclosed hereinare administered by inhalation into the lung of a subject who hasundergone lung transplantation. For further information on pulmonarydelivery of pharmaceutical compositions see Weiss et al., Human GeneTherapy 1999. 10:2287-2293; Densmore et al., Molecular therapy 1999.1:180-188; Gautam et al., Molecular Therapy 2001. 3:551-556; andShahiwala & Misra, AAPS PharmSciTech 2004. 24; 6(3):E482-6.Additionally, respiratory formulations for dsRNA are described in U.S.Patent Application Publication No. 2004/0063654. Respiratoryformulations for dsRNA are described in US Patent ApplicationPublication No. 2004/0063654. International Patent Publication No. WO2008/132723 to one of the assignees of the present invention, and herebyincorporated by reference in its entirety discloses therapeutic deliveryof dsRNA to the respiratory system.

The dosage of each therapeutic agent is determined independently.

Pharmaceutical compositions that include the nucleic acid moleculesdisclosed herein may be administered once daily (q.d.), twice a day(b.i.d.), three times a day (t.i.d.), four times a day (q.i.d.), or atany interval and for any duration that is medically appropriate.However, the therapeutic agent may also be dosed in dosage unitscontaining two, three, four, five, six or more sub-doses administered atappropriate intervals throughout the day. In that case, the nucleic acidmolecules contained in each sub-dose may be correspondingly smaller inorder to achieve the total daily dosage unit. The dosage unit can alsobe compounded for a single dose over several days, e.g., using a drugdelivery pump; or using a conventional sustained release formulationwhich provides sustained and consistent release of the dsRNAs over aseveral day period. Sustained release formulations are well known in theart. The dosage unit may contain a corresponding multiple of the dailydose. The composition can be compounded in such a way that the sum ofthe multiple units of a nucleic acids together contain a sufficientdose.

Pharmaceutical Compositions, Kits, and Containers

Provided are compositions, kits, containers and formulations thatinclude at least one therapeutic agents (e.g., small organic moleculechemical compound; protein, antibody, peptide, peptidomimetic andnucleic acid molecule) which target, decrease, down-regulate or inhibitthe expression/activity/function of the gene TLR2, for administering toa patient.

Also provided are compositions, kits, containers and formulations thatinclude at least two therapeutic agents (e.g., small organic molecule;protein, antibody, peptide, peptidomimetic and nucleic acid molecule),at least one therapeutic agent which target, decrease, down-regulate orinhibit the expression/activity/function of the gene TLR2 and at leastone therapeutic agent which target, decrease, down-regulate or inhibitthe expression/activity/function of the gene TLR4, for administering toa patient.

A kit may include at least one container and at least one label.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers can be formed from a variety of materialssuch as glass, metal or plastic. The container can hold amino acid(s),small molecule(s), nucleic acid(s), protein(s), peptides(s),peptidomimetic(s), cell population(s) and/or antibody(s). In oneembodiment, the container holds a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition describedherein and can have a sterile access port (for example the container canbe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle). The active agent in the composition canbe a nucleic acid molecule(s) capable of specifically binding TLR2and/or modulating the function of TLR2. The active agent in thecomposition can be a nucleic acid molecule(s) capable of specificallybinding TLR4 and/or modulating the function of TLR4. The active agentsin the composition can be a nucleic acid molecule(s) capable ofspecifically binding TLR2 and TLR4 and/or modulating the function ofTLR2 and TLR4.

Kits may further include associated indications and/or directions;reagents and other compositions or tools used for such purpose asdescribed herein.

A kit may further include a second container that includes apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and/or dextrose solution. It can further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, stirrers, needles, syringes, and/orpackage inserts with indications and/or instructions for use.

The units dosage ampules or multidose containers, in which thetherapeutic agents are packaged prior to use, may include anhermetically sealed container enclosing an amount of therapeutic agentor solution containing a therapeutic agent suitable for apharmaceutically effective dose thereof, or multiples of an effectivedose. The therapeutic agent is packaged as a sterile formulation, andthe hermetically sealed container is designed to preserve sterility ofthe formulation until use.

The container in which the therapeutic agent molecules are packaged maybe labeled, and the label may bear a notice in the form prescribed by agovernmental agency, for example the Food and Drug Administration, whichnotice is reflective of approval by the agency under Federal law, of themanufacture, use, or sale of the therapeutic material therein for humanadministration.

Federal law requires that the use of pharmaceutical compositions in thetherapy of humans be approved by an agency of the Federal government. Inthe United States, enforcement is the responsibility of the Food andDrug Administration, which issues appropriate regulations for securingsuch approval, detailed in 21 U.S.C. §301-392. Regulation for biologicmaterial, including products made from the tissues of animals isprovided under 42 U.S.C. §262. Similar approval is required by mostcountries. Regulations vary from country to country, but individualprocedures are well known to those in the art and the compositions andmethods provided herein preferably comply accordingly.

As such, provided herein is a pharmaceutical product which may include acombination of nucleic acid molecules in solution in a pharmaceuticallyacceptable injectable carrier and suitable for administration to apatient, and a notice associated with the container in a form prescribedby a governmental agency regulating the manufacture, use, or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofmanufacture, use, or sale of the solution comprising the nucleic acidsfor human administration. Compositions, kits and methods disclosedherein may include packaging a nucleic acid molecule disclosed hereinthat includes a label or package insert. The label may includeindications for use of the nucleic acid molecules such as use fortreatment or prevention of lung disorders or injury in a human,including treatment of acute respiratory distress syndrome (ARDS), acutelung injury, pulmonary fibrosis (idiopathic), bleomycin inducedpulmonary fibrosis, mechanical ventilator induced lung injury, chronicobstructive pulmonary disease (COPD), chronic bronchitis, emphysema,bronchiolitis obliterans after lung transplantation and lungtransplantation-induced acute graft dysfunction, and any other diseaseor conditions that are related to or will respond to down-regulation ofthe expression of TLR2 in a cell or tissue, alone or in combination incombination with other therapies; or to down-regulation of theexpression of TLR2 and TLR4, alone or in combination with othertherapies. A label may include an indication for use in reducingexpression of TLR2 gene. A label may include an indication for use inreducing expression of TLR2 gene and TLR4 gene. A “package insert” isused to refer to instructions customarily included in commercialpackages of therapeutic products, that contain information about theindications, usage, dosage, administration, contraindications, othertherapeutic products to be combined with the packaged product, and/orwarnings concerning the use of such therapeutic products, etc.

Those skilled in the art will recognize that other lung disorder/injurytreatments, drugs and therapies known in the art can be readily combinedwith the therapeutic combination disclosed herein and are hencecontemplated herein.

The methods and compositions provided herein will now be described ingreater detail by reference to the following non-limiting examples.

EXAMPLES Example 1 Generation of Sequences for Active dsRNA Compounds tothe TLR2 and TLR4 Genes and Production of the dsRNA Compounds

Using proprietary algorithms and the known sequence of the genesdisclosed herein, the antisense and corresponding sense sequences ofdsRNA compounds were generated. In addition to the algorithm, 20-, 21-,22-, and 23-mer oligomer sequences are generated by 5′ and/or 3′extension of the 19-mer sequences. The sequences that have beengenerated using this method are fully complementary to a segment ofcorresponding mRNA sequence.

SEQ IDs Numbers 5-12,136 provide oligonucleotide sequences useful in thepreparation of dsRNA compounds disclosed herein. Each sequence ispresented in 5′ to 3′ orientation.

For each gene there is a separate list of 19-mer sense and correspondingantisense oligonucleotide sequences, which are prioritized based ontheir score in the proprietary algorithm as the best sequences fortargeting the human gene expression.

The siRNA compounds disclosed herein are synthesized by any methodsdescribed herein, infra.

Example 2 Evaluation of Inhibitory Activity of dsRNA Compounds TargetingTLR2 and TLR4 Genes

Inhibitory activity of dsRNA compounds is assessed in vitro bytransfection of dsRNA compounds into human HeLa or human PC3 cells.

Preparation of Cells for dsRNA Transfection

HeLa cells (American Type Culture Collection) are cultured as describedin Czaudema, et al. (NAR, 2003. 31:670-82).

In each well of a 6-well plate, 1×105 human HeLa cells (ATCC, Cat#CCL-2)are inoculated in 2 mL growth medium in order to reach 30-50% confluenceone day later. Cells are then incubated in 37±1° C., 5% CO2 incubatorfor 24 hours. One day post inoculation, cell culture media is replacedwith 1.5 mL fresh growth medium per well.

dsRNA Transfection

Following incubation, cells are transfected with dsRNA compounds usingthe Lipofectamine™ 2000 reagent (Invitrogen) at final concentrationsranging between 0.0035 nM to 100 nM (final dsRNA concentration in cellculture wells). Cells are then incubated in a 37±1° C., 5% CO2 incubatorfor 48 hours.

For the determination of transfection efficiency, cells are similarlytransfected with a 20 nM solution of a Cy3-labeled dsRNA which targetsthe DDIT4 gene transcript.

As negative control, cells are similarly transfected with a scrambledsequence dsRNA (CNL_1) at final concentrations of 40 and 100 nM.

RNA Preparation for Real Time qPCR (qPCR)

At 48 h after transfection cells are harvested and RNA is extracted fromcells isolated using EZ-RNA kit [Biological Industries (#20-410-100)].

Transfection efficiency is tested by fluorescent microscopy.

Determining Inhibitory Activity In Vitro

The percent of down-regulation of gene expression using specific dsRNAcompounds disclosed herein is determined using qPCR analysis. Therelative quantity of target gene mRNA is determined using as templateRNA prepared from each of the dsRNA-transfected cell samples. dsRNAactivity is determined based on the ratio of the target gene mRNAquantity in dsRNA-treated samples versus non-transfected controlsamples.

Chemically modified dsRNA compounds disclosed herein are tested in vitroas described and are shown to down-regulate target gene expression.

Example 3 Stability of dsRNA Compounds

Nuclease resistance of the dsRNA compounds disclosed herein is tested inhuman serum and/or in bronchoalveolar lavage fluid (BALF).

For stability testing, a dsRNA compound is diluted in human serum or inbronchoalveolar lavage fluid (BALF) to a required final concentration(e.g. 7 μM). A 5 μL aliquot is transferred to 15 μL of 1.5×TBE-loadingbuffer, immediately frozen in liquid nitrogen, and transferred to −20°C. This represents “Time Point 0”. The remaining dsRNA solution isdivided into 5 μL aliquots, which are incubated at 37° C. for 30 min, 1h, 6 h, 8 h, 10 h, 16 h or 24 h.

Following incubation, dsRNA compound samples are transferred to 15 μL of1.5×TBE-loading buffer. 5 μL of each dsRNA compound in loading buffersample is loaded onto a non denaturing 20% polyacrylamide gel andelectrophoresis is performed. The positive control, double-strandmigration reference (a non-relevant, 19-base pairs, blunt-ended,double-stranded RNA with similar chemical modifications), andsingle-strand migration reference (a non-relevant ssRNA with chemicalmodifications), as well as the Time Point 0 sample are loaded on thesame gel and electrophoresed in parallel.

For dsRNA visualization the gel is stained with Ethidium bromidesolution (1.0 μg/μL).

Stability of dsRNA compounds disclosed herein is determined by examiningthe migration pattern of dsRNA samples on PAGE following incubation inhuman serum and/or in bronchoalveolar lavage fluid (BALF).

Example 4 Efficacy of dsRNA in Mouse Models of Orthotopic VascularizedAerated Lung Transplantation

Therapeutic efficacy of dsRNA compounds described herein in preventingprimary graft dysfunction caused by both prolonged cold ischemia andimmune rejection was tested in syngeneic and allogeneic mouse orthotopicmodels of lung transplantation. The method of orthotopic vascularizedaerated left lung transplantation in the mouse utilizes cuff techniquesfor the anastomosis of pulmonary artery, pulmonary veins and bronchus.This method has been reported in several publications (Okazaki et al.,Am J Transplant, 2007; 7:1672-9 and Krupnick et al. Nature Protocols,2009; vol. 4 No. 1:86-93).

dsRNA Test Compounds

One dsRNA compound targeting TLR4 (designated TLR4_4_S500) and two dsRNAcompounds targeting TLR2 (designated TLR2_7_S73 and TLR2_4_S73) weretested in syngeneic mouse orthotopic models of lung transplantation. OnedsRNA compound targeting TLR4 (designated TLR4_4_S500) and one dsRNAcompounds targeting TLR2 (designated TLR2_4_S73) were tested inallogeneic mouse orthotopic models of lung transplantation. A dsRNAcompound directed at enhanced green fluorescent protein (EGFP)(designated EGFP_5_S763) and/or vehicle (phosphate buffer solution(PBS)) served as negative control in these experiments.

Table 1 lists dsRNA compounds that were tested in syngeneic andallogeneic mouse orthotopic models of lung transplantation.

TABLE 1 DsRNA compound Target gene TLR4_4_S500 TLR4, toll-like receptor4 TLR2_7_S73 TLR2, toll-like receptor 2 TLR2_4_S73 TLR2, toll-likereceptor 2 EGFP_5_S763 EGFP, Enhanced green fluorescent protein

Table 2 provides the sense strand and the antisense strand sequences ofthe dsRNA compounds that were tested in syngeneic and allogeneic mouseorthotopic models of lung transplantation. Table 2 further provides thecross species data.

TABLE 2 DsRNA Antisense compound Sense 5′->3′ 5′->3′ cross speciesTLR4_4_S500 GAGUUCAGGUUAA UAUAUGUUAAC rat, mouse CAUAUA CUGAACUCTLR2_7_S73 GCAAACUGCGCAA AUUAUCUUGCG rat, mouse GAUAAU CAGUUUGCTLR2_4_S73 CCUCUUUGAAAUA UUUAAGUAUUU rat, mouse CUUAAA CAAAGAGGEGFP_5_S763 GGCUACGUCCAGG GGUGCGCUCCUGG 21-mer AGCGCACC ACGUAGCC

Table 3 provides the sense strand and the antisense strand modificationpatterns of the dsRNA compounds that were tested in syngeneic andallogeneic mouse orthotopic models of lung transplantation.

TABLE 3 DsRNA compound Sense 5′->3′ Antisense 5′->3′ TLR4_4_S500 2′-OMesugar modified ribonucleotides in 2′-OMe sugar modified ribonucleotidespositions: 2, 4, 6, 8, 10, 12, 14, 16 and 18 in positions: 1, 3, 5, 7,9, 11, 13, 15, 17 and 19 unmodified ribonucleotides in positions:unmodified ribonucleotides in positions: 1, 3, 5, 7, 9, 11, 13, 15, 17and 19 2, 4, 6, 8, 10, 12, 14, 16 and 18 3′-terminal phosphate3′-terminal phosphate TLR2_7_S73 2′-OMe sugar modified ribonucleotidesin 2′-OMe sugar modified ribonucleotides positions: 2, 4, 6, 8, 10, 12,14, 16 and 18 in positions: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19unmodified ribonucleotides in positions: unmodified ribonucleotides inpositions: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 2, 4, 6, 8, 10, 12, 14,16 and 18 No 3′-terminal phosphate No 3′-terminal phosphate TLR2_4_S732′-OMe sugar modified ribonucleotides in 2′-OMe sugar modifiedribonucleotides positions: 2, 4, 6, 8, 10, 12, 14, 16 and 18 inpositions: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 unmodifiedribonucleotides in positions: unmodified ribonucleotides in positions:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 2, 4, 6, 8, 10, 12, 14, 16 and 18No 3′-terminal phosphate No 3′-terminal phosphate EGFP_5_S763 2′-OMesugar modified ribonucleotides in 2′-OMe sugar modified ribonucleotidespositions: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 in positions: 1, 3, 5,7, 9, 11, 13, 15, 17, 19 and 21 unmodified ribonucleotides in positions:unmodified ribonucleotides in positions: 1, 3, 5, 7, 9, 11, 13, 15, 17,19 and 21 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 No 3′-terminal phosphateNo 3′-terminal phosphate

Dosage and Administration

dsRNA compounds were administered at the end of lung transplantationsurgery (immediately after anastomosis opening), by intratrachealinstillation to the recipient. The following doses of individual dsRNAcompounds were tested in these animal models: 6 μg/mouse, 12.5 μg/mouse,25 μg/mouse and 50 μg/mouse.

Mouse Syngeneic Lung Transplantation (C57B1/6->C57B1/6)

Experimental Design

Both donor and recipient were C57BL/6 mice. Prior to transplantationischemia reperfusion injury was induced by prolonged cold preservationof the lung transplant by 18 hours of cold storage in a low dextrosesolution with components similar to solutions used to preserve humanlung transplants (18 hours of cold ischemia time (CIT)). This methodinduced symptoms consistent with primary graft dysfunction 24 hourspost-transplantation. Within 5-10 minutes after reperfusion 25 μg/mouse(or a different dose as described herein) of siRNA specific for controlsiRNA, TLR2, TLR4 or both TLR2 and TLR4 was administered down thetrachea. Lung recipients were assessed 24 hours later for lung injury.

Administration

By intratracheal instillation of dsRNA solution to the lungs; 1 dose ofa dsRNA compound or of a combination of dsRNA compounds is administeredimmediately after anastomosis opening on Day 0.

Evaluation

Lung recipients were evaluated at 24 hours post transplantation throughassessing lung function, as measured by:

-   -   Gross pathology—appearance of pulmonary edema;    -   Pulmonary function in the post-transplanted lung—PaO2,        oxygenation of arterial blood in the left pulmonary artery;    -   Intra-airway accumulation of cellular infiltrates; and    -   Total amount and differential counts of bronchoalveolar lavage        (BAL) cells

Results

In this syngeneic model, mouse isografts exposed to prolonged coldischemia (18 hours CIT) develop impaired oxygenation, pulmonary edema,increased inflammatory cytokine production and intra-graft andintra-airway accumulation of granulocytes as measured 24 hourspost-transplantation. By contrast, mouse lung recipients of 1 hour coldpreserved grafts (1 hour CIT) had little evidence of lung injury 24hours post-transplantation.

Lung recipients that were treated with either dsRNA specific for TLR2 orwith a combination of both dsRNA specific for TLR2 and dsRNA specificfor TLR4 had significantly better function and significantly less BALcellular infiltrate, as compared to other treatment groups and to thenegative control animals (treated with vehicle or with dsRNA specificfor EGFP).

FIG. 1 (representative image of N=5/group) shows that combinedadministration of dsRNA specific for TLR2 (i.e. TLR2_4_S73), at a doseof 25 μg/mouse and dsRNA specific for TLR4 (i.e. TLR4_4_S500), at a doseof 25 μg/mouse, efficiently reduced pulmonary edema in this mouse modelof lung transplantation. No apparent edema was observed in any of thelungs treated with combination of dsRNA for TLR2 and dsRNA for TLR4.Similar results were obtained with a combination of TLR2_7_S73 andTLR4_4_S500 (with a dose of 25 μg/mouse of each). Similar results wereobtained with a dose of 12.5 μg/mouse of each of the TLR2 dsRNA compoundand TLR4 dsRNA compound (TLR2_7_S73 and TLR4_4_S500), while obviousedema appeared in animals that were treated with vehicle or with dsRNAtargeting EGFP).

FIG. 2 shows that impaired recipient pulmonary function, measured at 24hours after lung transplantation, was restored in mice treated with acombination of dsRNA specific for TLR2 and dsRNA specific for TLR4, aswell as in mice treated with a single dsRNA specific for TLR2, but notin mice treated with a single dsRNA directed at TLR4.

Two combinations of dsRNA specific for TLR2 and dsRNA specific for TLR4were tested in these experiments, at a ratio of 1:1:

-   -   (ii) a combination of 25 μg/mouse of each TLR2_7_S73 and        TLR4_4_S500, total therapeutic amount: 50 μg/mouse; and    -   (iii) a combination of 25 μg/mouse of each TLR2_4_S73 and        TLR4_4_S500, total therapeutic amount: 50 μg/mouse; and

Additional animal groups were tested with either individual dsRNAspecific for TLR2 or with an individual dsRNA specific for TLR4.

Two dsRNAs specific for TLR2 were tested in the experiments: TLR2_4_S73at a dose of 25 μg/mouse and TLR2_7_S73 at doses of 25 μg/mouse and 50μg/mouse.

One dsRNAs specific for TLR4 was tested in the experiments: TLR4_4_S500at doses of 25 μg/mouse and 50 μg/mouse.

Negative control animals were treated with vehicle.

The test article (composition comprising a combination of dsRNA TLR2 andTLR4 dsRNA; dsRNA specific for TLR2; dsRNA specific for TLR4; orvehicle) was administered immediately after opening of anastomosis andbeginning of reperfusion.

FIG. 2 shows that administration of dual target dsRNA composition(comprising TLR2_7_S73 and TLR4_4_S500 (N=5) or TLR2_4_S73 andTLR4_4_S500 (N=3)), at a dose of 25 μg/mouse of each of the dsRNAcompounds, significantly preserved pulmonary function, keeping bloodoxygenation at almost normal levels (PaO2=500-530 mm Hg).

Administration of a single dsRNA compound specifically targeting TLR2(TLR2_7_S73 (N=3) or TLR2_4_S73 (N=5)), at a dose of 25 μg/mouse of theindividual dsRNA compound, was also significantly effective inpreserving pulmonary function, keeping blood oxygenation at a levelsimilar to the level obtained for 1 hour CIT control group. Similarresults were obtained with a higher dose (50 μg/mouse) of a single dsRNAcompound specifically targeting TLR2 (TLR2_7_S73; (N=5)).

Significantly, similar results were obtained with two different dsRNATLR2 compounds (TLR2_7_S73 and TLR2_4_S73) that target different regionsof the TLR2 gene.

Administration of a single dsRNA compound specifically targeting TLR4(TLR4_4_S500), at doses of 25 μg/mouse (N=2) or 50 μg/mouse (N=3), wasnot effective in preserving pulmonary function, keeping bloodoxygenation at a level similar to the level obtained for the vehiclecontrol group.

FIG. 3 shows that impaired recipient pulmonary function, measured at 24hours after lung transplantation, was restored in mice treated with acombination of dsRNA specific for TLR2 and dsRNA specific for TLR4(identified in FIG. 3 as “siRNA cocktail”). A combination of TLR2_7_S73and TLR4_4_S500 was used in these experiments, at a ratio of 1:1. Threedoses were tested:

-   -   (i) a combination of 25 μg/mouse of each TLR2_7_S73 and        TLR4_4_S500, total therapeutic amount: 50 μg/mouse;    -   (ii) a combination of 12.5 μg/mouse of each TLR2_7_S73 and        TLR4_4_S500, total therapeutic amount: 25 μg/mouse; and    -   (iii) a combination of 6 μg/mouse of each TLR2_7_S73 and        TLR4_4_S500, total therapeutic amount: 12 μg/mouse

Negative control animals were treated with vehicle or with dsRNAspecific for EGFP (EGFP_5_S763) at a dose of 50 μg/mouse, 25 μg/mouse or12.5 μg/mouse.

The test article (composition comprising a combination of TLR2 dsRNA andTLR4 dsRNA; or vehicle; or dsRNA specific for EGFP (identified in FIG. 3as “control siRNA”)) was administered immediately after opening ofanastomosis and beginning of reperfusion.

FIG. 3 shows that following lung transplantation after 1 h of cold graftpreservation (a reperfusion control), pulmonary function is onlyslightly worsened (PaO2=363±31 mm Hg), however, prolongation of coldpreservation time (18 h CIT) leads to a dramatic reduction inrecipient's pulmonary function (PaO2=170±13 mm Hg for vehicle group),indicating severe PGD (grade 3; ISHLT definition). Administration ofdual target siRNA composition (comprising TLR2_7_S73 and TLR4_4_S500),at a dose of 25 μg/mouse of each of the dsRNA compounds, significantly(P<0.005) preserved pulmonary function (PaO2=435±64), keeping bloodoxygenation at almost normal levels (PaO2=500-530 mm Hg). Administrationof the same doses of non-targeting control dsRNA (EGFP_5_S763 at a doseof 50 μg/mouse) did not improve pulmonary function.

Administration of dual target dsRNA composition (comprising TLR2_7_S73and TLR4_4_S500), at a dose of 12.5 μg/mouse of each of the dsRNAcompounds, was also significantly effective (P<0.05) in preservingpulmonary function, keeping blood oxygenation at a level similar to thelevel obtained for 1 hour CIT control group. Administration of the samedoses of non-targeting control dsRNA (EGFP_5_S763 at a dose of 25μg/mouse) did not improve pulmonary function.

Administration of dual target dsRNA composition (comprising TLR2_7_S73and TLR4_4_S500), at a dose of 6 μg/mouse of each of the dsRNAcompounds, was not effective in preserving pulmonary function, keepingblood oxygenation at a level similar to the level obtained with vehicleand non-targeting control dsRNA (EGFP_5_S763 at a doses of 50 μg/mouse,25 μg/mouse and 12.5 μg/mouse), which did not improve pulmonaryfunction.

FIG. 4 shows that a combination of dsRNA specific for TLR2 and dsRNAspecific for TLR4 (TLR2_4_S73 and TLR4_4_S500), as well as an individualtreatment comprising dsRNA specific for TLR2 (TLR2_4_S73), diminishedintra-airway accumulation of granulocytes. One of the pathophysiologicalfeatures of PGD is rapid influx of cellular infiltrates to theinterstitial lung space, which is typically detected in patients' chestradiographs. Consistent with this, total bronchoalveolar lavage (BAL)cell counts in mice that underwent lung transplantation after 18 h ofCIT (vehicle group), were significantly (P<0.01) higher than those inmice that underwent lung transplantation after 1 h of CIT (24±6 vs 9±4cells×10̂5/lung respectively) (N=2). Treatment with a combination ofdsRNA specific for TLR2 and dsRNA specific for TLR4 (TLR2_4_S73 andTLR4_4_S500; (N=5)), at a dose of 25 μg/mouse of each of the dsRNAcompounds, as well as an individual treatment comprising dsRNA specificfor TLR2 (TLR2_4_S73), at a doses of 50 μg/mouse (N=2) or at a dose of25 μg/mouse (N=5), diminished cellular BAL infiltration associated withprolonged cold preservation. Moreover, treatment with a combination ofdsRNA specific for TLR2 and dsRNA specific for TLR4, as well as anindividual treatment comprising dsRNA specific for TLR2, diminishedgranulocyte (neutrophils, eosinophils, basophils) accumulation in thelung airways.

Administration of a single dsRNA compound specifically targeting TLR4(TLR4_4_S500), at a dose of 50 μg/mouse (N=2), was not effective indiminishing intra-airway accumulation of granulocytes, keepingintra-airway accumulation of granulocytes at a level similar to thelevel obtained with vehicle and non-targeting control dsRNA(EGFP_5_S763) at a dose of 50 μg/mouse (N=2), 25 μg/mouse (N=5) or 12.5μg/mouse (N=5).

Mouse Allogeneic Lung Transplantation (Balb/C->C57B1/6)

Experimental Design

In this model prolonged cold ischemia prevents lung allograft acceptancemediated by immunosuppression. In this model Balb/c lungs are subjectedto 18 hours of cold ischemia time (CIT) and are transplanted intoC57B1/6 recipients that are treated with immunosuppressants: anti-CD40Lon post operative day 0 and CTLA4Ig on day 2. In contrast to recipientswho received allografts stored for 1 hour, these stored for 18 hoursacutely rejected their allografts with marked intragraft accumulation ofIFNγ⁺ CD8 T cells.

Evaluation

Lung recipients were evaluated at 7 days post transplantation throughassessing:

-   -   Abundance of intragraft IFNγ+CD8+ T cells (by FACS)    -   Histopathological signs of acute graft rejection, A score

Administration

By intratracheal instillation of dsRNA solution to the lungs; 2 doses ofa dsRNA compound or of a combination of dsRNA compounds are administeredimmediately after anastomosis opening on Day 0 and on Day 1 post lungtransplantation.

Results

Administration of a combination of a dsRNA specific for TLR2 and dsRNAspecific for TLR4 (TLR2_4_S73 and TLR4_4_S500, identified as “siRNAcocktail”) with a dose of 25 μg/mouse of each of the dsRNA compounds(N=5), or of a single dsRNA specific for TLR2 (TLR2_4_S73), at a dose of25 μg/mouse (N=4), diminished abundance of intragraft IFNγ+CD8+ T cellsin allo-transplantation. In this allogeneic model, in prolonged coldischemia prevents lung allograft acceptance mediated byimmunosuppression. In this model Balb/c lungs are subjected to 18 hours(18 CIT) of cold ischemia and are transplanted into C57BL/6 (B6)recipients that are treated with anti-CD40L on postoperative day (POD) 0and CTLA4Ig on POD 2. Both of these immunosuppressive reagents arecurrently in pre-clinic development by major pharmaceutical companiesand when used together are generally referred to as double costimulatoryblockade treatment (DCB). In contrast to recipients who receivedallografts stored for 1 hour (1 CIT) (N=6−), 18 CIT Balb/c->DCB+B6 lungrecipients (N=6) acutely rejected their allografts with markedintragraft accumulation of IFNγ+CD8+T (FIG. 5 A, upper panel). Thisrejection was also evident by histopathological evaluation (FIG. 6 A,B).

In this model, control dsRNA (EGFP_5_S763) treated lung recipients (N=3)acutely rejected their allografts with significantly elevated IFNγ+CD8+T cells accumulation in allograft tissue. By contrast, recipient micetreated with a combination of a dsRNA specific for TLR2 and dsRNAspecific for TLR4 (TLR2_4_S73 and TLR4_4_S500, identified as “siRNAcocktail”; (N=5)) on days 0 and 1, had significantly decreased abundanceof intragraft IFNγ⁺ CD8⁺ T cells (FIG. 5 A, B), as well as significantlyless histological evidence of acute rejection (FIG. 6 A, B).

These experiments show that targeting TLR function using dsRNA compoundsspecific for TLR2 or a combination of dsRNA compounds specific for TLR2and TLR4 significantly improves/prevents lung graft injury. Lungfunction in TLR2 or TLR2- and TLR4-dsRNA treated recipients was similarto lung recipients of 1 hour cold preserved graft, indicating that thismethod may be useful in preventing/treating primary graft dysfunction inlung transplant recipients. These experimental procedures and dsRNAtreatments may be conducted in major histocompatibility complex(MHC)-mismatched donors and recipients.

Example 5 dsRNA Oligonucleotide Sense and Antisense Pairs

The Sequence Listing provides sense and antisense oligonucleotides forgenerating double-stranded oligonucleotide compounds, useful in carryingout the methods disclosed herein.

The sense and antisense strands of the TLR2 double-strandedoligonucleotides are provided in sense strand sequences set forth in SEQID NOs: 5-722; 1441-2246; 3053-4152; and 5253-5545 and antisense strandsequences set forth in SEQ ID NOs: 723-1440; 2247-3052; 4153-5252 and5546-5838.

The sense and antisense strands of the TLR4 double-strandedoligonucleotides are provided in sense strand sequences set forth in SEQID NOs: 5839-7075, 8313-8458, 8605-10318, 12033-12084 and antisensestrand sequences set forth in SEQ ID NOs: 7076-8312, 8459-8604,10319-12032, 12085-12136.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present disclosures teach oneskilled in the art to test various combinations described herein towardgenerating therapeutic combination with improved activity for treatinglung disorders or injury in a mammal. Such improved activity can includee.g., improved stability, improved bioavailability, improved activationof cellular responses mediating RNAi. Therefore, the specificembodiments described herein are not limiting and one skilled in the artcan readily appreciate that additional specific combinations can betested without undue experimentation toward identifying therapeuticcombinations with improved activity.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms “a” and“an” and “the” and similar referents in the context of describing theinvention (especially in the context of the following claims) are to beconstrued to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. The terms“comprising”, “having,” “including,” containing”, etc. shall be readexpansively and without limitation (e.g., meaning “including, but notlimited to,”). Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theinventions embodied therein herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and sub-generic groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1-169. (canceled)
 170. A method of treating a lung disorder, lungdisease, or lung injury associated with lung transplantation in asubject in need thereof, comprising administering to the subject atherapeutically-effective amount of at least one TLR2 inhibitor or apharmaceutically acceptable salt thereof, wherein the TLR2 inhibitorcomprises a nucleic acid molecule targeting a TLR2 gene, therebytreating the lung disorder, lung disease, or lung injury associated withlung transplantation in said subject.
 171. The method according to claim170, wherein the lung disorder associated with lung transplantation isselected from the group consisting of: inflammation, graft rejection,primary graft failure, ischemia-reperfusion injury, reperfusion injury,reperfusion edema, allograft dysfunction, acute graft dysfunction,pulmonary reimplantation response, bronchiolitis obliterans, and primarygraft dysfunction (PGD).
 172. The method according to claim 171, whereinthe lung disorder associated with lung transplantation is PGD.
 173. Themethod according to claim 172, wherein the treatment prevents or reducesthe symptoms of cold ischemia-associated PGD or warm ischemia-associatedPGD.
 174. The method according to claim 170, wherein the administrationis local administration.
 175. The method according to claim 174, whereinthe administration is by inhalation or by intratracheal instillation.176. The method according to claim 170, wherein the nucleic acidmolecule comprises a double-stranded oligonucleotide.
 177. The methodaccording to claim 176, wherein the double-stranded oligonucleotidecomprises: (a) a sense strand and an antisense strand; (b) each strandis independently 17 to 40 nucleotides in length; (c) a 17 to 40nucleotide sequence of the antisense strand is complementary to asequence of an mRNA encoding TLR2; and (d) a 17 to 40 nucleotidesequence of the sense strand is complementary to the antisense strand.178. The method according to claim 177, wherein the double-strandedoligonucleotide comprises a structure (A1): (A1) 5′ (N)x-Z 3′ (antisensestrand) 3′ Z′-(N′)y-z″ 5′ (sense strand)

wherein each of N and N′ is an unmodified ribonucleotide, a modifiedribonucleotide, or an unconventional moiety; wherein each of (N)x and(N′)y is an oligonucleotide in which each consecutive N or N′ is joinedto the next N or N′ by a covalent bond; wherein each of Z and Z′ isindependently present or absent, but if present is independently 1-5consecutive nucleotides or unconventional moieties or a combinationthereof covalently attached at the 3′ terminus of the strand in which itis present; wherein z″ may be present or absent, but if present is acapping moiety covalently attached at the 5′ terminus of (N′)y; whereineach of x and y is independently an integer between 17 and 40; whereinthe sequence of (N′)y is complementary to the sequence of (N)x; andwherein (N)x comprises an antisense sequence to an mRNA encoding TLR2having a sequence forth in SEQ ID NO:1.
 179. The method according toclaim 178, wherein x=y=19.
 180. The method according to claim 177,wherein the double-stranded oligonucleotide comprises a structure (A2):(A2) 5′ N1-(N)x-Z 3′ (antisense strand) 3′ Z′-N2-(N′)y-z″ 5′ (sensestrand)

wherein each of N2, N and N′ is independently an unmodifiedribonucleotide, a modified ribonucleotide, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond; wherein each of x and y is independently an integer between 17 and39; wherein the sequence of (N′)y is complementary to the sequence of(N)x and wherein (N)x is complementary to a consecutive sequence in anmRNA encoding TLR2; wherein N1 is covalently bound to (N)x and ismismatched to the mRNA encoding TLR2; wherein N1 is a moiety selectedfrom the group consisting of uridine, modified uridine, ribothymidine,modified ribothymidine, deoxyribothymidine, modified deoxyribothymidine,riboadenine, deoxyriboadenine, and modified deoxyriboadenine; wherein z″may be present or absent, but if present is a capping moiety covalentlyattached at the 5′ terminus of N2-(N′)y; and wherein each of Z and Z′ isindependently present or absent, but if present is independently 1-5consecutive nucleotides or unconventional moieties or a combinationthereof covalently attached at the 3′ terminus of the strand in which itis present.
 181. The method according to claim 180, wherein x=y=18. 182.The method according to claim 170, wherein the at least one TLR2inhibitor is administered to said subject prior to or during lungtransplantation.
 183. The method according to claim 170, wherein the atleast one TLR2 inhibitor is administered to said subject following lungtransplantation.
 184. The method according to claim 178, wherein one ofZ and Z′ is present.
 185. The method according to claim 178, whereinboth of Z and Z′ is present.
 186. The method according to claim 178,wherein wherein z″ is present.
 187. The method according to claim 180,wherein one of Z and Z′ is present.
 188. The method according to claim180, wherein both of Z and Z′ is present.
 189. The method according toclaim 180, wherein wherein z″ is present.